Activin type IIa receptor variants and pharmaceutical compositions containing the variants

Abstract

To provide extracellular activin type IIa receptor (ActRIIa) variants and pharmaceutical compositions containing these variants. [Solution] The polypeptide comprising the extracellular ActRIIa variant comprises the extracellular ActRIIa variant fused to a monomer or portion of the Fc domain. A pharmaceutical composition comprising the variant is used to treat diseases and conditions accompanied by muscle weakness and atrophy (e.g., Duchenne muscular dystrophy, faciocraniobrachial muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia), or metabolic diseases (e.g., obesity, type 1 diabetes, or type 2 diabetes).

Description

[Technical Field]

[0001] This invention relates to activin type IIa receptor variants and methods for using them. [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 involving motor neurons that control muscle weakness, muscle atrophy, and / or 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 extremities. IBM is an inflammatory muscle disease that primarily affects the muscles of the thighs as well as the muscles of the arms that control flexion of the fingers and wrists. ALS is a motor neuron disease characterized by generalized muscle rigidity, spasms, and atrophy due to degeneration of motor neurons. Efforts to improve the treatment and survival of subjects with these devastating muscle diseases have yet to be successful.

[0003] In the United States, overweight is a serious problem, affecting approximately 25% of the population. The fact that it increases both visceral and subcutaneous tissue leads to dysfunction of various organs. Overweight is a risk factor for a range of complications, including obesity, diabetes (e.g., type 1 and type 2 diabetes), cardiovascular disease, and several forms of cancer. Insulin resistance is also associated with obesity and occurs when pancreatic tissue requires high levels of insulin. When pancreatic beta cells can no longer produce enough insulin to meet their demands, hyperglycemia occurs, leading to the development of type 2 diabetes. The increased adipocytes in obese individuals 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, there are few treatment options available. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Novel treatments are needed for these muscle and metabolic diseases. [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-terminus or C-terminus of a monomer or portion of an Fc domain. Such portions may be linked by amino acids or other covalent bonds, thereby increasing 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 interactions between two Fc domain monomers. Polypeptides of the present invention may be used to increase muscle mass and strength in subjects having diseases or conditions including weakness and atrophy of muscles, such as Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The polypeptides of the present invention may also be used in subjects who have or are at risk of developing metabolic diseases (e.g., obesity, type 1 diabetes, or type 2 diabetes) to reduce body weight, reduce body fat, increase glucose clearance, enhance insulin sensitivity, or lower fasting insulin levels. Furthermore, the polypeptides of the present invention may also be used in subjects who are at risk of developing or have developed diseases or conditions involving muscle weakness and muscle atrophy, or metabolic diseases, to act on myostatin, activin, and / or bone morphogenetic protein 9 (BMP9) signaling.

[0006] In one aspect, the present invention is a polypeptide comprising an extracellular activin type IIa receptor (ActRIIa) variant, wherein the variant 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 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 has the sequence of VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 1), wherein in the sequence, X1 is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 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 from wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or from extracellular ActRIIa having one of the sequences of SEQ ID NOs: 76-96. Regarding polypeptides.

[0007] In some embodiments, the variants are 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(Sequence ID 2) The array has 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 It 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) having the sequence of, in the sequence, 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 are defined as described 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) having the sequence of, in the sequence, X2, X4, X7, X8, X9, X 14 、X 15 、X 16 、X 18 、X 22 、X 23 、X 25 、X 26 、and X 27 are defined as described above.

[0010] In some embodiments, the variant is GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14 HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS(Sequence ID 5) It has an array of X2, X4, X8, X9, X 14 , X 18 , X 23 , X 25 , X 26 , and X 27 It is defined as described above.

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

[0012] In any of the embodiments described above, the variant has one of the sequences from sequence numbers 6 to 72. In any of the embodiments described above, position X 24 The amino acids in this compound can be substituted with amino acid K.

[0013] In any of the embodiments described above, position X 24 The amino acids in this compound can be substituted with amino acid E. In any of the embodiments described above, the polypeptide 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 embodiments described herein, the polypeptide may further include a portion fused or covalently attached to the C-terminus of the polypeptide. In some embodiments, the portion increases the stability of the polypeptide or improves its pharmacokinetics. In some embodiments, the portion is an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin.

[0015] In any of the embodiments described herein, 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, the polypeptide comprising the extracellular ActRIIa variant described herein fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a 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 embodiments described herein, the polypeptides described herein may further include 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 includes one or more amino acid substitutions. In some embodiments, the Fc domain including one or more amino acid substitutions does not form a dimer.

[0017] In any of the embodiments described above, the polypeptide 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 embodiments described above, 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 embodiments described herein, the polypeptide 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. In some embodiments, the amino acid spacer is GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

[0021] In some embodiments, the amino acid spacer is GGGS (SEQ ID NO: 99), GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), SGGG (SEQ ID NO: 107), GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), G AGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117), GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), GGSGGSGGSGGS (SEQ ID NO: 123), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125) , GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127), GGGGAGGGGAGGGGA (SEQ ID NO: 128), GGGGSGGGGSGGGGS (SEQ ID NO: 129), AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139) These are GENLYFQSGG (sequence number 140), SACYCELS (sequence number 141), RSIAT (sequence number 142), RPACKIPNDLKQKVMNH (sequence number 143), GGSAGGSGSGSSGGSSGASGTGTAGTGSGSGTGSG (sequence number 144), AAANSSIDLISVPVDSR (sequence number 145), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (sequence number 146), EAAAK (sequence number 147), or PAPAP (sequence number 148).

[0022] In any of the embodiments described above, the polypeptides described herein have a serum half-life of at least 7 days. In any of the embodiments described above, the polypeptide described herein contains 200 pM or more of K D It binds to human bone morphogenetic factor 9 (BMP9). In some embodiments, this polypeptide binds to activin and / or myostatin, and its binding to human BMP9 is low (e.g., weak). In some embodiments, this polypeptide does not bind substantially to human BMP9.

[0023] In any of the embodiments described above, the polypeptide described herein has a K content of 800 pM or less. D It then binds to human activin A. In any of the embodiments described above, the polypeptide described herein has a K content of approximately 800 pM or less. D It then binds to human activin B.

[0024] In any of the embodiments described above, the polypeptide described herein contains approximately 5 pM or more of K D It then binds to human GDF-11. In another aspect, the present invention relates to nucleic acid molecules encoding polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants having any one sequence from SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)). In another aspect, the present invention also relates to vectors comprising nucleic acid molecules described herein.

[0025] In another embodiment, the present invention relates to a host cell expressing a polypeptide described herein, wherein the host cell comprises a nucleic acid molecule or vector described in the two embodiments, and the nucleic acid molecule or vector is expressed within the host cell.

[0026] In another aspect, the present invention relates to a method for producing a polypeptide as described herein, the method comprising: a) providing a host cell containing 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 enable the formation of the polypeptide.

[0027] In another aspect, the present 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 embodiment, the present invention also relates to a construct (e.g., a homodimer) comprising two identical polypeptides, each containing an extracellular ActRIIa variant having one of the sequences of Sequence IDs 1-72 (e.g., Sequence IDs 6-72) fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of Sequence ID No. 97). The two Fc domain monomers in these two polypeptides interact to form an Fc domain within the construct.

[0029] In another embodiment, the present invention also relates to a construct comprising two different polypeptides (e.g., heterodimers), each containing an extracellular ActRIIa mutant having one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), which is 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 embodiment, the present invention relates to a method for increasing muscle mass in a subject requiring such increase. This method comprises administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein, to the subject.

[0031] In some embodiments of methods for increasing the muscle mass of 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 embodiment, the present invention relates to a method for acting on myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their receptors) in subjects having a disease or condition including muscle weakness and muscle atrophy, wherein the method comprises administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject. In some embodiments of this embodiment, the disease or condition is DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia.

[0033] In another embodiment, the present invention relates to a method for treating a subject having DMD by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

[0034] In another embodiment, the present invention relates to a method for treating a subject having FSHD by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

[0035] In another embodiment, the present invention relates to a method for treating a subject having IBM by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

[0036] In another embodiment, the present invention relates to a method for treating a subject having ALS by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to a subject.

[0037] In another embodiment, the present invention relates to a method for reducing body fat in a subject requiring such reduction by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

[0038] In another embodiment, the present invention relates to a method for reducing the body weight of a subject in need by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

[0039] In another embodiment, the present invention relates to a method for lowering blood glucose levels in a subject requiring such treatment by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein.

[0040] In another embodiment, the present invention relates to a method for enhancing insulin sensitivity in a subject requiring such enhancement by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

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

[0042] In another embodiment, the present invention relates to a method for acting on myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their receptors) in subjects having or at risk of developing metabolic disorders, the method comprising administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the subject.

[0043] In another embodiment, the present invention relates to a method for treating and / or preventing a target metabolic disease by administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector, or a pharmaceutical composition, as described herein, to the target.

[0044] In some embodiments of any of the above embodiments, the metabolic disease is selected from the group including obesity, type 1 diabetes, and type 2 diabetes. In some embodiments of any of the above embodiments, the metabolic disease is obesity. In some embodiments of any of the above embodiments, the metabolic disease is type 1 diabetes. In some embodiments of any of the above embodiments, the metabolic disease is type 2 diabetes.

[0045] In some embodiments of the above embodiments, the method reduces the body weight and / or percentage of weight gain of the subject. In some embodiments of the above embodiments, the method reduces the amount and / or percentage of body fat of the subject. In some embodiments of the above embodiments, the method does not affect the subject's appetite for food intake. In some embodiments of the above embodiments, the method reduces obesity of the subject. In some embodiments of the above embodiments, the method reduces the weight of the epididymal and perirenal fat bodies of the subject. In some embodiments of the above embodiments, the method reduces the amount of subcutaneous fat and / or visceral fat of the subject. In some embodiments of the above embodiments, the method lowers the fasting insulin level of the subject. In some embodiments of the above embodiments, the method lowers the blood glucose level of the subject. In some embodiments of the above embodiments, the method increases the insulin sensitivity of the subject. In some embodiments of the above embodiments, the method increases the glucose clearance rate of the subject. In some embodiments of the above embodiments, the method improves the serum lipid profile of the subject. In some embodiments of the above embodiments, the method does not reduce lean body mass.

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

[0047] In some embodiments of any of the above-described aspects, a polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in sufficient quantities to increase muscle mass and / or strength, to act on myostatin, activin, and / or BMP9 signaling in a subject, or to reduce or inhibit the 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, alleviate obesity, reduce the weight of epididymal and perirenal fat bodies, reduce body fat percentage, reduce body weight, reduce the rate of weight gain, lower fasting insulin levels, lower blood glucose levels, increase insulin sensitivity, act on 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 serum lipid profile, regulate insulin biosynthesis and / or secretion from β-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 vascular complications (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 embodiments, the mutant has the sequence of SEQ ID NO: 69. In some embodiments, the mutant having the sequence of SEQ ID NO: 69 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 69 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0051] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 58. In some embodiments, the mutant having the sequence of SEQ ID NO: 58 is located at position X 17 amino acid K, position X 23 , X 24 , X25 , and X 26 The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 58 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0052] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 6. In some embodiments, the mutant having the sequence of SEQ ID NO: 6 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a mutant having a therapeutically effective amount of the sequence of SEQ ID NO: 6 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0053] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 38. In some embodiments, the mutant having the sequence of SEQ ID NO: 38 is at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 38 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0054] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 41. In some embodiments, the mutant having the sequence of SEQ ID NO: 41 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 41 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0055] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 44. In some embodiments, the mutant having the sequence of SEQ ID NO: 44 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 44 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0056] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 70. In some embodiments, the mutant having the sequence of SEQ ID NO: 70 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 70 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0057] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 71. In some embodiments, the mutant having the sequence of SEQ ID NO: 71 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 71 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0058] In some embodiments of any of the above embodiments, the mutant has the sequence of SEQ ID NO: 72. In some embodiments, the mutant having the sequence of SEQ ID NO: 72 is located at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26The amino acid sequence TEEN or TKEN and / or a C-terminal elongation (e.g., 1, 2, 3, 4, 5, 6 or more further amino acids at the C-terminus, e.g., amino acid NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method optionally includes a variant having a therapeutically effective amount of the sequence of SEQ ID NO: 72 at position X 17 amino acid K, position X 23 , X 24 , X 25 , and X 26 This includes administering a variant having the amino acid sequence TEEN or TKEN and / or a C-terminal extension, or a pharmaceutical composition containing the variant, to subjects in need of it (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), to act on myostatin, activin, and / or BMP9 signaling in subjects (e.g., subjects with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it), to reduce body fat or weight in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes), or to treat and / or prevent metabolic disorders in subjects (e.g., subjects with obesity, type 1 diabetes, or type 2 diabetes, or subjects at risk of developing it).

[0059] definition As used herein, the term “extracellular activin IIa receptor (ActRIIa) variant” refers to a peptide containing either the soluble extracellular portion of a one-pass transmembrane receptor ActRIIa having at least one amino acid substitution from wild-type extracellular ActRIIa (e.g., the bolded portion of the sequence in SEQ ID NO: 75 shown below) or one of the sequences in SEQ ID NOs: 76-96. 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] Extracellular ActRIIa mutants may have any one sequence from SEQ ID NOs: 1 to 72. In certain embodiments, extracellular ActRIIa mutants may have any one sequence from SEQ ID NOs: 6 to 72 (Table 2). In some embodiments, extracellular ActRIIa mutants may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity with the wild-type extracellular ActRIIa sequence (SEQ ID NO: 73).

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

[0064] Extracellular ActRIIb mutant (SEQ ID NO: 149): GRGEAETRECIFYNANWEKDRTNQSGLEPCYGDQDKRRHCFASWKNSSGTIELVKQGCWLDDINCYDRQECVAKKDSPEVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT As used herein, the term “linker” refers to a bond between two elements, for example, a peptide or protein domain. Polypeptides described herein may include extracellular ActRIIa variants fused to a portion (e.g., extracellular ActRIIa variants having any one of the sequences from SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). This portion can increase the stability of the polypeptide or improve its pharmacokinetic properties. This portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide via a linker. The linker may be a covalent bond or a spacer. The term “bond” refers to any type of bond resulting from a chemical bond, for example, an amide bond or a disulfide bond, or from a chemical reaction, for example, chemical conjugation. The term "spacer" refers to a portion (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids) present between two elements to provide space and / or flexibility between them. Amino acid spacers are part of the primary sequence of a polypeptide (e.g., fused to a peptide with space provided by the polypeptide backbone). For example, the formation of a disulfide bond between two hinge regions forming 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 is at least C H 2 domains and C H The Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100%) with a human Fc domain containing three domains. H 2 and C H3) includes. In some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not contain any portion of the immunoglobulin that can act as an antigen-recognition region, such as a variable domain or a complementarity-determining region (CDR). In the wild-type Fc domain, the two Fc domain monomers are two C H Dimerization occurs through interactions between the three antibody constant domains, as well as through one or more disulfide bonds formed between the hinge domains of the two dimer-forming Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack effector function, typical of a "dead Fc domain." In certain embodiments, each Fc domain monomer of the Fc domain is modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. H The antibody constant domain contains amino acid substitutions. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. The Fc domain may be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, such as 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 serum albumin and functions to bind to serum albumin. The albumin-binding peptide may be of different origins, such as 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, as well as to extracellular matrix components such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: P02751) having amino acids 610-702 of the sequence. 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 the blood. It accounts for approximately half of the serum proteins. In some embodiments, human serum albumin has the sequence UniProt ID NO: P02768 (SEQ ID NO: 154).

[0069] As used herein, the term “fused” is used to describe a combination or bond of two or more elements, components, or protein domains, such as peptides or polypeptides, by means including chemical conjugation, recombination, and chemical bonding, such as amide bonding. For example, two single peptides can be fused in series via chemical conjugation, chemical bonding, peptide linkers, or any other covalent bonding means to form a single continuous protein structure, such as a polypeptide. In some embodiments of the polypeptides described herein, an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence from SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) may be fused in series to the N-terminus or C-terminus of a certain 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 dimerization), 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)) via a linker. For example, an extracellular ActRIIa mutant is fused to a certain region (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a peptide linker. In this case, the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIa mutant via a chemical bond, such as a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of that region (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a chemical bond, such as 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 containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of the sequences from SEQ ID NOs. 1–70 (e.g., SEQ ID NOs. 6–70)). A C-terminal extension may consist of 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 two-amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NOs. 155) (a six-amino acid C-terminal extension). Any amino acid sequence that does not interfere with the polypeptide activity may be used. SEQ ID NOs. 71, i.e., the sequence of SEQ ID NOs. 69 having the C-terminal extension of NP, and SEQ ID NOs. 72, i.e., the sequence of SEQ ID NOs. 69 having the C-terminal extension of NPVTPK, represent two possible ways in which the polypeptide of the present invention may 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 of a candidate sequence, e.g., an extracellular ActRIIa variant, that are identical to the amino acid (or nucleic acid) residues of a reference sequence, e.g., wild-type extracellular ActRIIa (e.g., SEQ ID NO: 73), after the sequences have been aligned and gaps have been introduced as necessary to obtain the maximum percentage identity (i.e., gaps may be introduced in either or both the candidate sequence and the reference sequence for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for determining the percentage identity can be achieved in various ways within the scope of the art, such as using publicly available computer software, e.g., BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for evaluating the alignment, including algorithms necessary to obtain the maximum alignment over the entire length of the sequences to be compared. In some embodiments, the percentage of amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (or, this can also be expressed as a given candidate sequence having or containing a particular percentage of amino acid (or nucleic acid) sequence identity with respect to a given reference sequence) is calculated as follows: 100 × (Fraction of A / B) In the formula, A is the number of amino acid (or nucleic acid) residues that are given the same score in the alignment of the candidate sequence and 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 percentage of amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence will not be equal to the percentage of 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 with a candidate sequence may demonstrate that the candidate sequence exhibits 50% to 100% identity over its entire length or a selected portion of consecutive amino acid (or nucleic acid) residues. The length of the candidate sequence aligned for comparison 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 the positions of the candidate sequences are occupied by the same amino acid (or nucleic acid) residues as the corresponding positions in the reference sequence, then those molecules are identical at those positions.

[0073] As used herein, the term “serum half-life” refers to the time it takes for the plasma concentration of a therapeutic protein in a subject to be reduced by half, in the context of administering the therapeutic protein to that subject. Proteins may be redistributed, eliminated from the bloodstream, or degraded, for example, by proteolysis. Polypeptides containing extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence from SEQ ID NOs. 1–72 (e.g., SEQ ID NOs. 6–72)) exhibit a serum half-life of 7 days in humans, as described herein.

[0074] As used herein, the term “metabolic disorder” refers to a disease, disorder, or syndrome related to the metabolism of the subject, such as breaking down carbohydrates, proteins, and fats in food to release energy, and converting chemicals into other substances and transporting them into cells for energy utilization and / or storage. Some symptoms of metabolic disorders 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 disorders increase the risk of developing other diseases, such as cardiovascular disease. In this invention, metabolic disorders include, but are not limited to, obesity, type 1 diabetes, and type 2 diabetes.

[0075] As used herein, the term "weight gain rate (%)" refers to the percentage increase in weight compared to the subject's previous weight at a previous point in time. The weight gain rate (%) can be calculated as follows:

[0076] 100 × [(Weight at a later point in time - Weight at an earlier point in time) / (Weight at an earlier point in time)] In the present invention, administration to a subject of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), a nucleic acid molecule encoding an extracellular ActRIIa variant comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence 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 percentage of weight gain (%) of the subject.

[0077] As used herein, the term “appetite for food intake” refers to the subject’s natural desire or need for food. The subject’s appetite for food intake can be monitored by measuring the amount of food consumed after administration of a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1–72 (e.g., SEQ ID NOs. 6–72)). In the present invention, administration of a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1–72 (e.g., SEQ ID NOs. 6–72)), a nucleic acid molecule encoding a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1–72 (e.g., SEQ ID NOs. 6–72)), or a vector containing such a nucleic acid molecule to a subject does not affect the subject’s appetite for food intake.

[0078] As used herein, the term “adiposity” means the fat stored in the adipose tissue of the subject. In the present invention, administration to a subject of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), a nucleic acid molecule encoding an extracellular ActRIIa variant comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence 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 obesity without affecting lean body mass.

[0079] As used herein, the term “lean mass” refers to components of body composition, including, for example, lean mass, body fat, and body fluids. Typically, lean mass is calculated by subtracting the weight of body fat and body fluids from total body weight. Typically, the lean mass of a subject is between 60% and 90% of total body weight. In the present invention, administering to a subject a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), a nucleic acid molecule encoding an extracellular ActRIIa variant containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence 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 obesity (i.e., fat) without affecting lean mass.

[0080] As used herein, the term “epididymal and perirenal fat pads” refers to the densely packed adipocytes in the epididymis and perirenal region. In the present invention, administration to a subject of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence 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 sequence 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 subject's epididymal and perirenal fat pads.

[0081] As used herein, the term “fasting insulin” refers to the insulin level of a subject during a period of time (i.e., 12–24 hours) when the subject has not consumed any food. Fasting insulin levels are used to diagnose metabolic disorders. Fasting insulin levels are also used as an indicator of whether a subject is at risk of developing a metabolic disorder. Typically, in subjects with type 1 diabetes, the subject’s fasting insulin level is lower than that of a healthy subject. In subjects with insulin resistance (i.e., type 2 diabetes), the subject’s fasting insulin level is higher than that of a healthy subject. In the present invention, administration of a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), a nucleic acid molecule encoding a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), or a vector containing such a nucleic acid molecule to a subject 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 the blood. Glucose clearance rate can be measured in a glucose tolerance test (GTT). In a GTT, a subject is administered a fixed amount of glucose, and then a blood sample is taken to determine how quickly it is removed from the blood. Glucose clearance rate can be used as a parameter in 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 a profile of serum lipids. A "lipid profile" refers to the measurement of the distribution of different types of lipids and lipoproteins in the serum of a subject. Such measurements can be achieved through a set of blood tests. The types of lipids and lipoproteins in the serum of a subject include, but are not limited to, cholesterol (e.g., high-density lipoprotein (HDL) and low-density lipoprotein (LDL)), triglycerides, and free fatty acids (FFA). The distribution of different types of lipids and lipoproteins can be used as parameters in diagnosing and / or determining the risk of developing metabolic diseases such as obesity, diabetes, and insulin resistance. High levels of cholesterol, and especially low-density lipoproteins, 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 a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), a nucleic acid molecule encoding a polypeptide containing an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence of SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)), or a vector containing such a nucleic acid molecule to a subject improves the subject's serum lipid profile to the extent that cholesterol (particularly low-density lipoprotein) and triglyceride levels are reduced.

[0084] As used herein, the terms “affinity” or “binding affinity” refer 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 expressed using the dissociation constant (K). D ) or affinity constant (K A ) is described by. Two molecules with low binding affinity to each other generally tend to bind slowly and dissociate easily, and large K D This indicates that two molecules with high affinity for each other generally tend to bind easily and maintain the bond for a longer period, and a small K D This shows the K of the two interacting molecules. D This can be determined, for example, using methods and techniques well known in the art, such as surface plasmon resonance. D is, k off / k on It is calculated as a ratio.

[0085] As used herein, the term "muscle mass" refers to a component of body composition. Muscle mass is typically calculated by subtracting the weight of body fat and body fluids from total body weight. The percentage of muscle mass can vary significantly between individuals depending on their genetic makeup, age, race, and health status. Typically, a person's muscle mass may range from 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, polypeptides comprising extracellular ActRIIa variants as 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). Polypeptides of the present invention comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence from SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) have a weak binding affinity to BMP9 (e.g., K D It may have a concentration of 200 pM or higher.

[0087] As used herein, the term “vascular complication” refers to any vascular disorder or damage to a blood vessel, such as damage to the vessel wall. Damage to the vessel wall can lead to increased vascular permeability or leakage. The term “vascular permeability or leakage” refers to the vessel wall’s ability to allow small molecules, proteins, and cells to flow into and out of a blood vessel. Increased vascular permeability or leakage can result from an increase in the gaps between endothelial cells lining the vessel wall (e.g., an increase in the size and / or number of gaps) and / or thinning of the vessel wall.

[0088] As used herein, the term “polypeptide” refers to a single polymer in which monomers are amino acid residues covalently linked to each other via amide bonds. Polypeptides include any amino acid sequence, whether native, 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 may form a homodimer by 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, the polypeptides described herein, including an extracellular ActRIIa variant fused to an Fc domain monomer, may form a homodimer through the interaction of the two Fc domain monomers, where these Fc domain monomers 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 may form a heterodimer by covalent or non-covalent bonds. For example, the polypeptides described herein, including extracellular ActRIIa variants fused to Fc domain monomers, may form a heterodimer through the interaction of two Fc domain monomers fused to different ActRIIa variants, where these Fc domain monomers form the Fc domain in the heterodimer.

[0091] As used herein, the term “host cell” refers to a medium containing the necessary cellular components, such as organelles required to express proteins from the corresponding nucleic acids. The nucleic acids are generally contained within nucleic acid vectors that can be introduced into host cells by prior art known methods (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection). The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a mammalian cell (e.g., CHO cells or HEK293 cells).

[0092] As used herein, the term “therapeutic dose” means an amount of the polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition containing the polypeptide, nucleic acid, or vector of the present invention, that is effective in achieving a desired therapeutic effect in treating patients with diseases such as muscle diseases, or diseases or conditions accompanied by muscle weakness and muscle atrophy, such as Duchenne muscular dystrophy (DMD), faciocraniobrachial muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The term “therapeutic dose” also means an amount of the polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition containing the polypeptide, nucleic acid, or vector of the present invention, that is effective in achieving a desired therapeutic effect in treating diseases such as metabolic diseases, or conditions accompanied by excess body weight, excess body fat, hyperglycemia, elevated fasting insulin levels, or insulin resistance, such as obesity, type 1 diabetes, or type 2 diabetes. In particular, a therapeutic dose of polypeptide, nucleic acid, or vector avoids harmful side effects.

[0093] As used herein, the term “pharmaceutical composition” refers to a pharmaceutical or pharmaceutical preparation comprising an active ingredient and excipients and diluents to make the active ingredient suitable for a method of administration. The pharmaceutical compositions of the present invention comprise a polypeptide, nucleic acid, or pharmaceutically acceptable component compatible with a vector. The pharmaceutical composition may be in the form of a tablet or capsule for oral administration, or in an aqueous dosage 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 the other components of the formulation and must not be harmful to the recipient. In this invention, a pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutically stable to a polypeptide containing an extracellular ActRIIa variant, a nucleic acid molecule encoding the polypeptide, or a vector containing such a nucleic acid molecule. The properties of the carrier or excipient vary depending on the mode of administration. For example, aqueous carriers are generally used for intravenous administration, while solid carriers are preferred for oral administration.

[0095] As used herein, the term “treat and / or prevent” means treating and / or preventing a disease, such as a metabolic disease (e.g., obesity, type 1 diabetes and type 2 diabetes) or a muscular disease (e.g., DMD, FSHD, IBM, and ALS), using the methods and compositions of the present invention. Generally, treatment of a metabolic or muscular disease is performed after a subject has developed a metabolic or muscular disease and / or after a subject has already been diagnosed with a metabolic or muscular disease. Prevention of a metabolic or muscular disease refers to a step or procedure taken when a subject is at risk of developing a metabolic or muscular disease. A subject may exhibit signs or mild symptoms that a physician determines to be indicative of or a risk factor for developing a metabolic or muscular disease, or that a physician determines to be indicative of having a family history or a genetic predisposition to developing such a metabolic or muscular disease, even if the subject has not yet developed such a disease.

[0096] As used herein, the term “subject” refers to mammals, for example, preferably humans. Mammals include, but are not limited to, humans and domesticated and farm animals, such as monkeys (e.g., cynomolgus macaques), mice, dogs, cats, horses, and cattle. [Effects of the Invention]

[0097] According to the present invention, activin type IIa receptor mutants and pharmaceutical compositions containing the mutants can be provided. [Brief explanation of the drawing]

[0098] [Figure 1] Figure 1 shows sequence alignments of the wild-type sequences of extracellular ActRIIa and ActRIIb, as well as amino acid substitutions in the ActRIIa mutant. [Figure 2A] Figures 2A and 2B are scatter plots showing the effect of the extracellular ActRIIa mutant on body weight. Mice received a single hydrodynamic injection of either the plasmid construct encoding the shown ActRIIa mutant or a control plasmid. [Figure 2B] Figures 2A and 2B are scatter plots showing the effect of the extracellular ActRIIa mutant on body weight. Mice received a single hydrodynamic injection of either the plasmid construct encoding the shown ActRIIa mutant or a control plasmid. [Figure 3A] Figures 3A and 3B are bar graphs showing the effect of the extracellular ActRIIa mutant on muscle mass. [Figure 3B] Figures 3A and 3B are bar graphs showing the effect of the extracellular ActRIIa mutant on muscle mass. [Figure 4A] Figure 4A is a scatter plot showing the effect of the extracellular ActRIIa mutant on body weight. Mice received intraperitoneal injections of either the purified recombinant ActRIIa mutant or the vehicle control twice a week for four weeks. [Figure 4B] Figure 4B is a bar graph showing the effect of the extracellular ActRIIa mutant on individual muscle weight based on tissue analysis. [Figure 5A] Figure 5A is a scatter plot showing the effect of the extracellular ActRIIa mutant on body weight during the course of the experiment. Mice received a single hydrodynamic injection of either the plasmid construct encoding the indicated ActRIIa mutant or a control plasmid. [Figure 5B] Figure 5B is a bar graph showing the effect of the extracellular ActRIIa mutant on body weight at the end of day 28. [Figure 6A] Figures 6A and 6B are bar graphs showing the effect of the extracellular ActRIIa mutant on body weight based on tissue analysis. [Figure 6B] Figures 6A and 6B are bar graphs showing the effect of the extracellular ActRIIa mutant on body weight based on tissue analysis. [Figure 7A] Figures 7A and 7B are scatter plots showing the effects of different doses of extracellular ActRIIa mutants on body weight. Mice received intraperitoneal injections of either the purified recombinant ActRIIa mutant or the vehicle control twice a week for four weeks. [Figure 7B] Figures 7A and 7B are scatter plots showing the effects of different doses of extracellular ActRIIa mutants on body weight. Mice received intraperitoneal injections of either the purified recombinant ActRIIa mutant or the vehicle control twice a week for four weeks. [Figure 8A] Figures 8A and 8B are bar graphs showing the effects of different doses of extracellular ActRIIa mutants on muscle mass (Figure 8A) and fat mass (Figure 8B). [Figure 8B] Figures 8A and 8B are bar graphs showing the effects of different doses of extracellular ActRIIa mutants on muscle mass (Figure 8A) and fat mass (Figure 8B). [Figure 9A] Figures 9A and 9B are bar graphs showing the effect of different doses of extracellular ActRIIa mutants on muscle mass based on tissue analysis. [Figure 9B] Figures 9A and 9B are bar graphs showing the effect of different doses of extracellular ActRIIa mutants on muscle mass based on tissue analysis. [Modes for carrying out 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 having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIa variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) via interactions between two Fc domain monomers. The ActRIIa variants described herein have weaker or no binding affinity to bone morphogenetic factor 9 (BMP9) compared to activin and myostatin. The present invention also includes methods for treating diseases and conditions involving muscle weakness and muscle atrophy by increasing muscle mass and strength in a subject by administering polypeptides comprising the extracellular ActRIIa variant described herein, methods for treating or preventing metabolic diseases, or methods for acting on myostatin, activin, and / or BMP9 signaling.

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

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

[0102] There are two types of activin type II receptors: ActRIIa and ActRIIb. According to research, BMP9 has been shown to bind to ActRIIb with a binding affinity approximately 300 times that of ActRIIa (see, for example, Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIa is known to have a longer half-life compared to ActRIIb. The present invention describes an extracellular ActRIIa variant constructed by introducing amino acid residues of ActRIIb into ActRIIa for the purpose of conferring the physiological properties provided by ActRIIb while maintaining the beneficial physiological and pharmacokinetic properties of ActRIIa. Optimal peptides retain a longer serum half-life and, for example, a low binding affinity for BMP9 while providing a significant increase in muscle mass. Preferred ActRIIa variants also show improved binding to activin and / or myostatin compared to wild-type ActRIIa, whereby they can compete with endogenous activin receptors with respect to ligand binding and can 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 a decrease in body fat, body weight, or insulin resistance (e.g., an increase in insulin sensitivity). In some embodiments, amino acid substitutions can be introduced into the extracellular ActRIIa variant to reduce or eliminate the binding affinity of the variant for BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIa and ActRIIb are shown below.

[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 variants 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 different positions of 27 sites can be introduced into the extracellular ActRIIa variant (Table 1). 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). 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., the extracellular ActRIIa variant having the sequence of SEQ ID NO: 1) may include amino acid substitutions at all 27 positions as listed in Table 1. In some embodiments, the extracellular ActRIIa variant may include amino acid substitutions at a plurality of positions, e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 positions, among the 27 positions as listed in Table 1.

[0104] The 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, position X of the sequences shown in Tables 1 and 2 (SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) 17It is important that the lysine (K) group is retained. Substitution at that position can lead to loss of activity. For example, GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS(Sequence ID 150) ActRIIa mutants having the sequence X exhibit reduced activity in vivo. 17 This indicates that substitution of lysine (K) with alanine (A) in position X is not permitted. Therefore, the mutants in Tables 1 and 2 (e.g., ActRIIa mutants of the present invention, including SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72)) are at position X 17 It retains amino acid K.

[0105] The ActRIIa variant of the present invention preferably has reduced, weak, or substantially absent binding to BMP9. Position X 23 , X 24 , X 25 , and X 26 ActRIIa mutant containing the amino acid sequence TEEN and position X 24 Maintain amino acid K, and position X 23 , X 24 , X 25 , and X 26 In mutants having the amino acid sequence TKEN, BMP9 binding is reduced. Sequences TEEN and TKEN are interchangeable in the ActRIIa mutants of the present invention (e.g., the mutants in Tables 1 and 2, e.g., SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72)) to result in reduced BMP9 binding.

[0106] The ActRIIa variants of the present invention may further include a C-terminal elongation (e.g., additional amino acids at the C-terminus). The C-terminal elongation can have 1 to 6 additional amino acids (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) added 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 elongation that may be included in the ActRIIa variants of the present invention is the amino acid sequence NP. For example, a sequence containing a C-terminal elongation is SEQ ID NOs. 71 (e.g., SEQ ID NOs. 69 having C-terminal elongation NP). Another example of a C-terminal elongation that may be included in the ActRIIa variants of the present invention is the amino acid sequence NPVTPK (SEQ ID NOs. 155). For example, a sequence containing a C-terminal elongation is SEQ ID NOs. 72 (e.g., SEQ ID NOs. 69 having C-terminal elongation NPVTPK).

[0107] [Table 1]

[0108] In some embodiments of the extracellular ActRIIa mutant having the sequence of Sequence ID No. 2, X3 is E, X6 is R, and X 11 is D, X 12 is K, and X 13 is R, and X 16 is K or R, X 17 is K, and X 19 is W, and X 20 L is X 21 is D, and X 22 is I or F. In some embodiments of extracellular ActRIIa mutants having sequence number 1 or 2, X 17 It is K. In some embodiments of extracellular ActRIIa mutants having sequences of sequence numbers 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 extracellular ActRIIa mutants having any one sequence from sequence numbers 1 to 5, X 17 is K, and X 23 is T, and X 24 is K, and X 25 is E, and X 26 It is N.

[0109] In some embodiments, the polypeptides described herein include extracellular ActRIIa variants having any one of the sequences 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, the 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)), is located at position X 17 It contains amino acid K. Position X 17 Changing the amino acids results in a decrease in activity. For example, GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS(Sequence ID 150) ActRIIa mutants having the sequence X exhibit reduced activity in vivo. 17 This shows that substitution of K with A is not permitted.

[0115] In some embodiments, position X 23 , X 24 , X 25 , and X 26 The polypeptide of the present invention, comprising an extracellular ActRIIa mutant having the sequence TEEN at position X (for example, any one of SEQ ID NOs. 1 to 72 (for example, SEQ ID NOs. 6 to 72)), 24 It may have a substitution of amino acid E with amino acid K. In some embodiments, position X 23 , X 24 , X 25 , and X 26 The polypeptide of the present invention, comprising an extracellular ActRIIa mutant having the sequence TKEN at position X (for example, any one of SEQ ID NOs. 1 to 72 (for example, SEQ ID NOs. 6 to 72)), 24 It may have a substitution of amino acid K with amino acid E at position X. 23 , X 24 , X 25 , and X 26 Polypeptides having the sequences TEEN or TKEN exhibit reduced or weak binding to BMP9.

[0116] In some embodiments, the 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 having 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 having the C-terminal extension NPVTPK). One to six additional amino acids (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) can be added to the C-terminus of the C-terminal extension.

[0117] In some embodiments, the 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 having an amino acid substitution (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin), and this moiety 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 a heterodimer) via the interaction between two Fc domain monomers, and these Fc domain monomers combine to form an Fc domain in the dimer.

[0118] In some embodiments, the extracellular ActRIIa variants 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 of at least 7 days in humans. The polypeptides have a K content of 200 pM or higher. D It can bind to bone morphogenetic factor 9 (BMP9). This polypeptide has a K content of 10 pM or higher. D It can bind to activin A. In some embodiments, this polypeptide does not bind to BMP9 or activin A. In some embodiments, this polypeptide binds to activin and / or myostatin and shows low (e.g., weak) binding to BMP9. In some embodiments, polypeptides with low or weak binding to BMP9 are located at position X 23 , X 24 , X 25 , and X 26 It has the sequence TEEN or TKEN.

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

[0123] II. Fc domain In some embodiments, the polypeptides described herein may include extracellular ActRIIa variants fused to an Fc domain monomer or fragment of an Fc domain of an immunoglobulin in order to extend the serum half-life of the polypeptide. Polypeptides containing extracellular ActRIIa variants fused to an Fc domain monomer can form dimers (e.g., homodimers or heterodimers) via interactions between two Fc domain monomers, where these Fc domain monomers form an Fc domain in the dimer. As is conventionally known in the art, the Fc domain is a protein structure found at the C-terminus of immunoglobulins. The Fc domain is C HThe antibody comprises two Fc domain monomers that form a dimer through interaction between the three antibody constant domains. The wild-type Fc domain forms the minimal structure that binds to Fc receptors, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, typically in "dead" Fc domains. For example, the Fc domain may contain specific amino acid substitutions known to minimize interaction between the Fc domain and the 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 the homology region of the antibody sequence with a "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 containing 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 an Fc domain monomer that can be fused to an 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, the extracellular ActRIIa variants described herein (e.g., extracellular ActRIIa variants having any one sequence from SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97) by conventional genetic or chemical means, e.g., chemical coupling. 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-terminus or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant.

[0124] In some embodiments, the polypeptides described herein may include extracellular ActRIIa variants fused to an Fc domain. In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. In some embodiments, the Fc domain includes 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, such as a recombinant Fc domain.

[0125] Methods for producing Fc domains with reduced dimerization are known in the art. In some embodiments, C is used to inhibit dimerization due to steric collisions. H 3-C H 3. One or more amino acids having large side chains (e.g., tyrosine or tryptophan) may be introduced at the dimer interface. In other embodiments, C is introduced to eliminate favorable interactions. H 3-C H 3. One or more amino acids having small side chains (e.g., alanine, valine, or threonine) may be introduced at the dimer interface. H Methods for introducing amino acids having large or small side chains into three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication No. 2006 / 0074225, U.S. Patent 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, C between two Fc domains H 3-C H C that constitutes the 3 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 specific charged amino acid. HMethods for introducing charged amino acids into 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 present invention, the Fc domain is subjected to the following amino acid substitutions relative to the human IgG1 sequence: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, The Fc domain includes one or more of 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 particular embodiment, the Fc domain includes the amino acid substitution T366W relative to the human IgG1 sequence. The sequence of the wild-type Fc domain is shown in SEQ ID NO: 151.

[0128] III. Albumin-binding peptides In some embodiments, the polypeptides described herein may include extracellular ActRIIa variants fused to serum protein-binding peptides. Binding to serum protein peptides can improve the pharmacokinetics of protein drugs.

[0129] As an 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 extracellular ActRIIa mutants, an albumin-binding peptide can be ligated to the N-terminus or C-terminus (e.g., C-terminus) of an extracellular ActRIIa mutant described herein (e.g., an extracellular ActRIIa mutant having any one sequence from SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)). In some embodiments, the albumin-binding peptide is ligated directly or via a linker to the N-terminus or C-terminus of the extracellular ActRIIa mutant.

[0131] In some embodiments, the extracellular ActRIIa variants described herein (e.g., extracellular ActRIIa variants having any one sequence from 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, e.g., chemical coupling. Optionally, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the albumin-binding peptide. While not theoretically bound, it is conceivable that the inclusion of an albumin-binding peptide in the extracellular ActRIIa variants described herein could result in extended retention of the therapeutic protein via its binding to serum albumin.

[0132] IV. Fibronectin Domain In some embodiments, the polypeptides described herein may include extracellular ActRIIa variants fused to a fibronectin domain. Binding to a fibronectin domain can improve the pharmacokinetics of protein drugs.

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

[0134] As an example, fibronectin domains usable in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: P02751) having amino acids 610-702 of the sequence. In another embodiment, the fibronectin domain is an adnectin protein.

[0135] In some embodiments, the extracellular ActRIIa variants described herein (e.g., extracellular ActRIIa variants having any one sequence from 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, such as chemical coupling. Optionally, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the fibronectin domain. While not theoretically limited, it is conceivable that the inclusion of a fibronectin domain in the extracellular ActRIIa variants described herein could lead to 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 include extracellular ActRIIa variants fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of the protein drug.

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

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

[0139] In some embodiments, the extracellular ActRIIa variants described herein (e.g., extracellular ActRIIa variants having any one sequence from 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, e.g., chemical coupling. Optionally, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and human serum albumin. While not theoretically bound, it is thought that the inclusion of human serum albumin in the extracellular ActRIIa variants described herein may result in extended retention of the therapeutic protein.

[0140] VI. Linker The polypeptides described herein may include extracellular ActRIIa variants fused to a portion via a linker (e.g., extracellular ActRIIa variants having any one sequence from SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72)). In some embodiments, the portion enhances the stability of the polypeptide. Examples of portions include Fc domain monomers, wild-type Fc domains, Fc domains with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), albumin-binding peptides, fibronectin domains, 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 dimerization), an albumin-binding peptide (e.g., SEQ ID NO: 152), a fibronectin domain (e.g., SEQ ID NO: 153), or human serum albumin (e.g., SEQ ID NO: 154)) and an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence from SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) may 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 spacers may include motifs of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 98), GGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 100), GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), or SGGG (SEQ ID NO: 107), such as multiple or repeating motifs.In some embodiments, the spacer may contain 2 to 12 amino acids including the motifs of GA or GS, for example, GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer may contain 3 to 12 amino acids including the motifs of GGA or GGS, for example, GGA, GGS, GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). In some embodiments, the spacer may contain 4 to 12 amino acids including the motifs of GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), for example, GGAGGGAG (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 may contain the motifs of GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), for example, GGGGAGGGGAGGGGA (SEQ ID NO: 128) and GGGGSGGGGSGGGGS (SEQ ID NO: 129).In some embodiments of the present invention, the amino acid spacer between a portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or serum albumin) and an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence from SEQ ID NOs. 1 to 72 (e.g., SEQ ID NOs. 6 to 72)) may be GGG, GGGA (SEQ ID NOs. 98), GGGG (SEQ ID NOs. 100), GGGAG (SEQ ID NOs. 130), GGGAGG (SEQ ID NOs. 131), or GGGAGGG (SEQ ID NOs. 132).

[0141] In some embodiments, the spacer may also include amino acids other than glycine, alanine, and serine, such as AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146). In some embodiments, the spacer may include the motif EAAAK (Sequence ID 147), for example, a multiple or repeating motif. In some embodiments, the spacer is (XP) n (where X may be any amino acid (e.g., A, K, or E), and n is 1 to 5), and may include proline-rich sequence motifs such as PAPAP (SEQ ID NO: 148), e.g., multiple or repeating motifs.

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

[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 medium containing the necessary cellular components, such as organelles required to express the polypeptides and fusion polypeptides described herein from the corresponding nucleic acids. These nucleic acids may be contained within nucleic acid vectors that can be introduced into host cells by prior art known methods (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, or infection). The choice of nucleic acid vector depends 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 The nucleic acid sequence encoding the amino acid sequence of the polypeptide of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. The nucleic acid molecule encoding the polypeptide of the present invention can be obtained, for example, using standard techniques such as gene synthesis. Alternatively, the nucleic acid molecule encoding wild-type extracellular ActRIIa can be mutated to include specific amino acid substitutions, for example, using standard techniques in the art such as QuikChange® mutagenesis. The nucleic acid molecule 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 prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may contain various components, which can be tuned and optimized to suit a particular host cell. For example, vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding the 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 fetal 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 (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. Examples of E. coli strains, though not limited to them, include 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 of protein products (e.g., glycosylation). A suitable cell line or host system can be selected to ensure proper modification and processing of the expressed polypeptide. The expression vector can be introduced into a suitable host cell using prior art methods, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector has been introduced into the host cell for protein production, the host cell is cultured in a conventional nutrient medium modified as needed to induce a promoter, select transformants, or amplify the gene encoding the desired sequence.Methods for expressing therapeutic proteins are known in the art, and refer to, for example, Paulina Balbas and 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 The host cells used to produce the polypeptides of the present invention are known in the art and can be grown in a culture medium suitable for 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 the necessary addition of a selected drug, such as ampicillin. The 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 of 5-10%. The pH of the medium is generally about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. The expression vector of the present invention uses 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., a mammalian host cell) into a cell culture medium. Protein recovery may involve filtering the cell culture medium to remove cellular residue. 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, e.g., chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. The proteins can be isolated and purified by appropriately selecting an affinity column, such as a protein A column (e.g., POROS protein A chromatography), and combining it 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 proteins. Once the cells are disrupted, the cellular residue can be removed by centrifugation or filtration. In some cases, polypeptides can be conjugated to marker sequences such as peptides to facilitate purification. An example of a marker amino acid sequence is the hexahistidine peptide (His tag), which binds to an agarose affinity column with nickel functional groups at micromolar affinity. 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 polypeptide of the present invention may be used, for example, in gene therapy, in vectors containing nucleic acid molecules encoding the polypeptide of the present invention (e.g., viral vectors (e.g., retroviral vectors, adenovirus vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutant vaccinia ankara (MVA: Modified) The protein can be produced by target cells (e.g., human) by administering Vaccinia Ankara, adeno-associated virus vectors, and alphavirus vectors. If the vector is present in the target cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it promotes polypeptide expression, which is then secreted from the cells. If the desired outcome is the treatment of a disease or disorder, no further action is likely required. If protein collection is desired, blood can be collected 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 a pharmaceutical composition comprising a polypeptide as described herein (for example, a polypeptide comprising an extracellular ActRIIa variant (for example, an extracellular ActRIIa variant having any one sequence from SEQ ID NOs. 1 to 72 (for example, SEQ ID NOs. 6 to 72))). In some embodiments, the pharmaceutical composition of the present invention comprises a polypeptide comprising an extracellular ActRIIa variant having a C-terminal elongation (for example, 1, 2, 3, 4, 5, 6 or more additional amino acids) (for example, an extracellular ActRIIa variant having any one sequence from SEQ ID NOs. 1 to 70 (for example, SEQ ID NOs. 6 to 70)) as a therapeutic protein. In some embodiments, the pharmaceutical composition of the present invention comprises a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence from SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a certain moiety (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or serum albumin) as a therapeutic protein. In some embodiments, the pharmaceutical composition of the present invention comprising the polypeptide of the present invention can be used in combination in therapy with other agents (e.g., therapeutic biologics and / or small molecules) or compositions. In addition to a therapeutically effective amount of polypeptide, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers or excipients, which can be prepared by methods known to those skilled in the art. In some embodiments, the pharmaceutical composition of the present invention comprises a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding the polypeptide of the present invention, or a vector containing such a nucleic acid molecule.

[0152] The acceptable carriers and excipients of the pharmaceutical composition are non-toxic to the recipient at the dose and concentration used. Acceptable carriers and excipients may include buffers such as phosphates, citrates, 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 injectable formulation. The injectable pharmaceutical composition 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, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). Preparation methods are well 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 prepared in microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the present invention may also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and This is described in Practice of Pharmacy, 22nd edition (2012). Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0154] The pharmaceutical composition of the present invention may also be prepared as a sustained-release formulation. A preferred example of a sustained-release formulation is a semipermeable matrix of a solid hydrophobic polymer containing the polypeptide 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 allow for the release of molecules over several months, for example, 1 to 6 months, while other formulations release the pharmaceutical composition of the present invention over a shorter period, for example, several days to several weeks.

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

[0156] The pharmaceutical composition for gene therapy may be in an acceptable diluent or may comprise a slow-release matrix in which a gene delivery vehicle is embedded. When hydrodynamic injection is used as the delivery method, the pharmaceutical composition containing a nucleic acid molecule encoding the polypeptide described herein or a vector containing that nucleic acid molecule (e.g., a viral vector) is readily delivered intravenously in a large volume. Vectors that can be used as in vivo gene delivery vehicles include, but are not limited to, retroviral vectors, adenovirus 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 A pharmaceutical composition containing the polypeptide of the present invention as a therapeutic protein may be formulated for, for example, intravenous, parenteral, subcutaneous, intramuscular, intra-arterial, subarachnoid, or intraperitoneal administration. The pharmaceutical composition may also be formulated for orally, nasally, nebulized, aerosolically, rectally, or vaginally, or administered via these methods. With regard to injectable formulations, various effective pharmaceutical carriers are known in the art. For example, see the ASHP Handbook on Injectable Drugs, Toissel, 18th edition (2014).

[0158] In some embodiments, a pharmaceutical composition comprising a nucleic acid molecule encoding the polypeptide of the present invention or a vector containing such a nucleic acid molecule can be delivered by gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors usable for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenovirus vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutant vaccinia ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the mRNA molecule encoding the polypeptide of the present invention may be administered directly to the target.

[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. Hydrodynamic injection places the nucleic acid molecules encoding the polypeptides described herein under the control of a potent promoter within a manipulated plasmid (e.g., a viral plasmid). Plasmids are often readily delivered intravenously in large volumes. Hydrodynamic injection uses controlled hydrodynamic pressure intravenously to enhance cellular permeability, resulting from the high pressure obtained from rapid injection of large volumes of fluid, which leads to extravascular extrusion of the fluid and plasmid from the vein. Nucleic acid molecule expression is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injection of 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 is determined by factors including the route of administration, the disease being treated, and the physical characteristics of the subject, such as age, weight, and health status. The pharmaceutical composition of the present invention may contain the polypeptide of the present invention in a dosage 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), in more specific embodiments, about 0.1 to about 30 mg / kg, and in more specific embodiments, about 0.3 to about 30 mg / kg. The dosage can be adapted by a physician according to conventional factors such as the severity of the disease and various parameters.

[0161] Pharmaceutical compositions are administered in a therapeutically effective dose to bring about improvement or restoration of symptoms, in a form appropriate to their dosage form. Pharmaceutical compositions are administered in various dosage forms, such as intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Generally, therapeutic proteins are administered at doses of 0.1 to 100 mg / kg, for example, 1 to 50 mg / kg. Pharmaceutical compositions containing the polypeptide of the present invention can be administered to subjects requiring it, for example, daily, weekly, bi-weekly, monthly, bi-monthly, quarterly, semi-annually, annually, or once or more as medically required (e.g., 1 to 10 times or more). In some embodiments, pharmaceutical compositions containing the polypeptide of the present invention can be administered to subjects requiring it weekly, bi-weekly, monthly, bi-monthly, or quarterly. Dosages can be provided in a single or multiple-dose regimen. The timing between doses can be reduced as the medical condition improves or increased as the patient's health deteriorates.

[0162] X. Treatment method This invention is based on the discovery that substituting amino acids from the extracellular portion of ActRIIb to the extracellular portion of ActRIIa yields ActRIIa mutants with improved properties. ActRIIa mutants created by introducing residues from ActRIIb to ActRIIa retain beneficial properties of ActRIIa, such as a longer serum half-life and lower binding affinity to 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 mutant properties create useful therapeutic properties that can compete with endogenous activin receptors in terms of ligand binding. Because ActRIIa mutants contain the extracellular portion of the receptor, they are soluble and can bind to and sequester ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Therefore, extracellular ActRIIa mutants 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, myostatin loss 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 could be a viable approach to increasing muscle mass. Indeed, the extracellular ActRIIa mutants of the present invention increase muscle mass in mice (see Examples 1-3 and 5-6). These data demonstrate that the extracellular ActRIIa mutants described herein can increase muscle mass and can be used to treat subjects with diseases or conditions resulting in muscle weakness or muscle atrophy.

[0163] Furthermore, these data provide compelling reasons why the extracellular ActRIIa mutants of the present invention can be used to treat other diseases or conditions associated with increased expression of activin receptor or activin receptor ligands, such as metabolic diseases (e.g., obesity, type 1 diabetes, and type 2 diabetes). Numerous studies have shown that increasing muscle mass is one way to reduce body fat and / or body weight, which suggests that metabolic diseases (e.g., obesity, type 1 diabetes, and type 2 diabetes) can be indirectly treated by increasing muscle mass using the extracellular ActRIIa mutants described herein. However, since increased activin receptor and activin receptor ligands have been shown in obese mice and humans, obesity can be treated using the extracellular ActRIIa mutants described herein by reducing the increase in activin receptor signaling (e.g., by binding to endogenous activin receptor ligands, e.g., activin and myostatin, and sequestering them).

[0164] The present invention provides compositions and therapeutic methods that can be used to increase muscle mass and strength in subjects requiring it. In some embodiments, subjects may have a disease that causes muscle weakness or muscle atrophy (e.g., muscle weakness or atrophy of skeletal muscle). In some embodiments, the methods described herein relate to acting on myostatin, activin, and / or BMP9 signaling in subjects having a disease or condition involving muscle weakness and muscle atrophy. In some embodiments, polypeptides comprising the extracellular ActRIIa variant 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, the target muscle mass is increased by 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))).

[0165] In some embodiments, polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))) may be administered to a subject to increase muscle mass or to act on 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 involving muscle weakness and muscle atrophy (e.g., Duchenne muscular dystrophy (DMD), faciocraniobrachial muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS) sarcopenia, or cancer cachexia).

[0166] The present invention also includes a method for treating subjects having Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia by administering to a subject a polypeptide described herein (for example, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence 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 and type 2 diabetes). In some embodiments, the subject may have a disease that causes obesity. In some embodiments, the methods described herein relate to acting on myostatin, activin, and / or BMP9 signaling in subjects having obesity, diabetes (type 1 and type 2 diabetes), or a disease or condition that causes obesity. In some embodiments, polypeptides comprising the extracellular ActRIIa variant 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, by 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))), a reduction in the subject's body fat (e.g., body fat mass or body fat percentage), a reduction in the subject's body weight or weight gain, a decrease in fasting insulin levels, an increase in glucose clearance, or an increase in insulin sensitivity (e.g., a decrease in insulin resistance).

[0168] In some embodiments, polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))) may be administered to a subject to prevent the development of obesity (e.g., in patients at risk of developing obesity (e.g., overweight patients, patients with a family history of obesity, or patients with 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 extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72)) to a subject may help reduce the subject's weight by reducing the amount of fat. In some embodiments, extracellular ActRIIa variants reduce the amount of fat while maintaining or increasing lean body mass.

[0169] In some embodiments, polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))) may be used to prevent the development of diabetes (e.g., type 1 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 disorders) may be prophylactically administered polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))) so that the extracellular ActRIIa polypeptides may maintain the normal function and healthy state of β-cells and prevent or delay autoimmune inflammatory damage to β-cells. In other embodiments, polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))) may be administered to an individual before diagnosis of diabetes (e.g., type 1 and type 2 diabetes) or before diagnosis of clinical symptoms of diabetes (e.g., hyperglycemia, high fasting insulin levels, insulin resistance, polyuria, polydipsia, polyphagia). In some embodiments, extracellular ActRIIa polypeptides can be administered to a patient before the patient requires insulin. In yet another embodiment, administration of extracellular ActRIIa polypeptides can delay or postpone the need for insulin therapy in diabetic patients. For example, administration of the extracellular ActRIIa polypeptides of the present invention to a subject may help increase the rate of glucose clearance from the blood.

[0170] In some embodiments, polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence 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 development of obesity or diabetes (e.g., type 1 and type 2 diabetes), or to act on myostatin, activin, and / or BMP9 signaling (e.g., to reduce or inhibit the 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 obesity, reduce the weight of epididymal and perirenal fat bodies, or reduce body fat percentage). 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 method described herein improves the serum lipid profile of the subject. In some embodiments, the method described herein reduces body fat without reducing lean body mass (e.g., without affecting or increasing lean body mass). In some embodiments, the method described herein reduces body fat and increases muscle mass. In some embodiments, the method described herein lowers blood glucose levels (e.g., fasting blood glucose levels) and / or increases glucose clearance. In some embodiments, the method described herein lowers fasting insulin levels and / or improves insulin sensitivity (e.g., reduces insulin resistance). In some embodiments, the method described herein modulates insulin biosynthesis and / or secretion from β-cells. In some embodiments, the method described herein does not affect appetite for food intake.In some embodiments, the methods described herein do not cause vascular complications in subjects such as increased vascular permeability or leakage.

[0171] In some embodiments, polypeptides described herein (e.g., polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having any one sequence of SEQ ID NOs. 1-72 (e.g., SEQ ID NOs. 6-72))) reduce body fat, reduce body weight, or increase 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 any one sequence of SEQ ID NOs. 1-71 (e.g., SEQ ID NOs. 6-71)) and further comprising a C-terminal extension of 1-6 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., homodimer or heterodimer) of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one sequence 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 dimerization), 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 such nucleic acids, may also be administered according to any of the methods described herein. In any of the methods described herein, polypeptides, nucleic acids, or vectors may be administered as part of a pharmaceutical composition. [Examples]

[0173] Example 1: Effect of extracellular ActRIIa mutant on body weight C57Bl / 6 mice were subjected to a single hydrodynamic injection of a plasmid construct encoding one of the following six polypeptides. Injection was administered (n=10 / group).

[0174] (1) Human Fc (hFc), (2) Extracellular ActRIIa (SEQ ID NO: 73) fused to the N-terminus of hFc via the GGG linker, (3) Extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via the GGG linker, (4) Extracellular ActRIIa mutant (SEQ ID NO: 69) fused to the N-terminus of hFc via the GGG linker, and (5) Extracellular ActRIIb mutant (SEQ ID NO: 149) fused to the N-terminus of hFc via a GGG linker.

[0175] A 100 μg plasmid construct was delivered over 5–8 seconds at a volume equivalent to 10% of body weight. 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 potent and ubiquitous promoter. The protein of interest was then secreted and freely circulated under the hepatocyte's endogenous machinery. Mice were weighed twice weekly for 30 days, and measurements were recorded as absolute body weight (BW) in grams and as a percentage change in body weight from baseline (Figures 2A and 2B, respectively).

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

[0177] (1) Human Fc (hFc), (2) Extracellular ActRIIa (SEQ ID NO: 73) fused to the N-terminus of hFc via the GGG linker, (3) Extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via the GGG linker, (4) Extracellular ActRIIa mutant (SEQ ID NO: 69) fused to the N-terminus of hFc via the GGG linker, and (5) Extracellular ActRIIb mutant (SEQ ID NO: 149) fused to the N-terminus of hFc via a GGG linker.

[0178] A 100 μg plasmid construct was delivered over 5–8 seconds at a volume equivalent to 10% of body weight. High-volume, short-duration injection provided the necessary pressure to introduce the plasmid into hepatocytes where it would be expressed, particularly ensuring the target protein was expressed under a potent and ubiquitous promoter. The target protein was then secreted under the hepatocyte's endogenous mechanisms and circulated freely. On days 0 (baseline), 14, and 28 of the study, mice were subjected to NMR analysis using a MiniSpec LF90 NMR analyzer (Bruker, Woodlands, Texas) to determine lean body mass. The percentage change in lean body mass from baseline was recorded on days 14 and 28 (Figures 3A and 3B).

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

[0180] (1) Tris-buffered saline, (2) Extracellular ActRIIa (SEQ ID NO: 73) fused to the N-terminus of hFc via the GGG linker, (3) Extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via the GGG linker, (4) Extracellular ActRIIa / b mutant (SEQ ID NO: 69) fused to the N-terminus of hFc via the GGG linker, (5) Extracellular ActRIIa / b9Δ9 mutant (SEQ ID NO: 58) fused to the N-terminus of hFc via the 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 proteins were generated by transient expression in HEK293 cells and purified from conditioned medium using protein A Sepharose chromatography. After four weeks of administration, mice were humanely sacrificed and necropsied. Necropsies included collecting weights of the whole body, as well as the gastrocnemius, pectoralis, and quadriceps muscles. Statistical analysis of muscle / body weight data was performed using GraphPad Prism 7 (GraphPad Software, La Jolla, California) (Figures 4A and 4B, respectively).

[0182] Example 4: Evaluation of ActRIIa mutant binding affinity by surface plasmon resonance (SPR) Biacore 3000 was used to measure the dynamics of interactions between the ActRIIa mutant and its ligands: activin A, activin B, growth factor 11 (GDF11), and BMP-9. The ActRIIa mutant was expressed and purified according to the method described in Example 3. The ActRIIa mutant was immobilized on a tip (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 from GE Healthcare® was used as the running buffer. Each ligand was flowed at a constant concentration system at 40 μl / min to avoid mass transfer effects. The K of each interaction was measured. D To calculate this, the data was analyzed using Scrubber2 with BioLogic™ software (Table 4).

[0183] [Table 4]

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

[0185] (1) Vehicle, (2) pLEV113-ActRIIa(19~127) (SEQ ID NO: 73) fused to the N-terminus of hFc via the GGG linker, (3) pLEV113-ActRIIb(41~155) (SEQ ID NO: 74) fused to the N-terminus of hFc via the GGG linker, (4) pLEV113-ActRIIa / b (SEQ ID NO: 69) fused to the N-terminus of hFc via the GGG linker, (5) pLEV113-ActRIIb / a (SEQ ID NO: 149) fused to the N-terminus of hFc via the GGG linker, (6) pLEV113-ActRIIa / b+ (SEQ ID NO: 150) fused to the N-terminus of hFc via the GGG linker, (7) pLEV113-ActRIIa / b-delta 9m2 (SEQ ID NO: 38) fused to the N-terminus of hFc via the GGG linker, (8) pLEV113-ActRIIa / b-delta 9m3 (SEQ ID NO: 41) fused to the N-terminus of hFc via the GGG linker, (9) pLEV113-ActRIIa / b-delta 9m4 (SEQ ID NO: 44) fused to the N-terminus of hFc via the 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 the GGG linker, and (12) pLEV113-ActRIIa / bmax1 (SEQ ID NO: 72) fused to the N-terminus of hFc via a GGG linker.

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

[0187] Example 6: Dose effect of extracellular ActRIIa mutant on body weight, muscle weight, and muscle mass. Eight-week-old male C57BL / 6 mice were weight-matched into nine groups (n=10 / group). Each group was administered either 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 evaluated doses were 20 mg / kg, 8 mg / kg, 3 mg / kg, and 1 mg / kg. Treatment was administered intraperitoneally (IP) twice a week for four weeks (8 doses), and the study was completed on day 28 of the study. During the study, body weight was recorded on the days of administration (Figures 7A and 7B). At the end of the study, the groups were NMR-imaged for mass spectrometry of lean body mass and fat (Figures 8A and 8B), and muscle weights of the pectoralis and gastrocnemius muscles 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 body-matched treatment groups (n=10 / group). All animals were maintained on either a standard solid diet (Chow; Purina LabDiet5001 (St. Louis, Missouri)) or a high-fat diet (HFD; Research Diets® D12331 (New Brunswick, New Jersey)). The solid diet group and the HFD group were further divided into groups receiving either the ActRII mutant or a vehicle twice weekly for 60 days. Body weight was measured twice weekly at treatment. Body composition was measured using MiniSpec LF50 at baseline (before administration of the treatment and transition to HFD), and then every other week until the end of the study. On the end of the study, the tissues of interest (serum, plasma, muscle, and fat deposits) were surgically removed and weighed. Next, serum samples were evaluated for obesity biomarkers, and plasma was evaluated for HbA1c levels.

[0189] Other embodiments While the present invention has been described in relation to its specific embodiments, further modifications are possible, and this application is intended to encompass any variations, uses, or adaptations of the present invention that generally adhere to the principles of the present invention. It will be understood that any such deviation from this disclosure may fall within the scope of known or relevant practices within the art to which the present invention belongs and may constitute the essential features described above.

[0190] All publications, patents, and patent applications are incorporated herein by reference in the same way as when each individual publication, patent, and patent application is specifically and individually indicated as being incorporated herein by reference in the entirety of that publication.

[0191] Other embodiments are also within the scope of the following claims. The technical concepts that can be understood from the above embodiments are described below as an addendum. [Note 1] Includes extracellular activin type IIa receptor (ActRIIa) mutant, The aforementioned variant 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 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 The sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 1) is present, where 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; X 10 is K or Q; X 11 is D; X 12 is K; X 13 is R and 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 Q is a polypeptide.

[0192] [Note 2] X1 is F, and X 10 The polypeptide is K, as described in Appendix 1. [Note 3] The polypeptide according to Appendix 1, wherein the mutant has one of the sequences from sequence numbers 6 to 72.

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

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

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

[0196] [Note 9] A vector containing the nucleic acid molecule specified in Appendix 8. [Note 10] A pharmaceutical composition comprising a polypeptide described in any one of the appendices 1 to 7, a nucleic acid molecule described in appendice 8, or a vector described in appendice 9, and one or more pharmaceutically acceptable carriers or excipients.

[0197] [Note 11] A pharmaceutical composition for treating a subject having a disease or condition including muscle weakness or muscle atrophy, wherein the pharmaceutical composition comprises a therapeutically effective amount of a polypeptide described in any one of Appendix 1 to 7, a nucleic acid molecule described in Appendix 8, or a vector described in Appendix 9, and the pharmaceutical composition is administered to the subject.

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

[0199] [Note 13] A pharmaceutical composition for increasing muscle mass in a subject requiring it, wherein the pharmaceutical composition comprises a therapeutically effective amount of a polypeptide described in any one of Appendix 1 to 7, a nucleic acid molecule described in Appendix 8, or a vector described in Appendix 9, and the pharmaceutical composition is administered to the subject.

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

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

[0202] [Note 16] Includes extracellular activin type IIa receptor (ActRIIa) variants, the variants of which are 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 The sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 1) is present, where X1 is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 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 The molecule is E or Q, and the variant is a polypeptide having at least one amino acid substitution from wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or from extracellular ActRIIa having one of the sequences of SEQ ID NOs: 76-96.

[0203] [Note 17] The above 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 The polypeptide described in Appendix 16, having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 2).

[0204] [Note 18] The above variant is GAILGRSETQECLFX2NANWEX4X5RTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 The polypeptide described in Appendix 16 or 17, having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 3).

[0205] [Note 19] The above variant is GAILGRSETQECLFX2NANWEX4DRTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 KX 25 X 26 PX 27 A polypeptide having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 4), as described in any one of the appendices 16 to 18.

[0206] [Note 20] The above variant is GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 A polypeptide having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 5), as described in any one of the appendices 16 to 19.

[0207] [Note 21] The polypeptide described in Note 16, wherein X1 is F. [Note 22] The polypeptide described in Note 16, wherein X1 is Y. [Note 23]X 10 A polypeptide as described in Appendix 16, 21, or 22, wherein is K.

[0208] [Note 24]X 10 A polypeptide as described in Appendix 16, 21, or 22, wherein Q is Q. [Note 25] A polypeptide according to any one of the items in Notes 16 to 24, wherein X2 is F. [Appendix 26] A polypeptide according to any one of Appendix 16 to 24, wherein X2 is or Y.

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

[0210] [Note 29] A polypeptide according to any one of the items in Notes 16 to 28, wherein X4 is K. [Note 30] A polypeptide according to any one of the notes 16 to 28, wherein X4 is L. [Appendix 31] A polypeptide according to any one of the appendices 16, 17, 18, and 21-30, wherein X5 is D.

[0211] [Appendix 32] A polypeptide according to any one of the appendices 16, 17, 18, and 21-30, wherein X5 is E. [Appendix 33] A polypeptide according to any one of Appendices 16, 17, and 21-32, wherein X6 is R.

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

[0213] [Appendix 36] A polypeptide according to any one of Appendices 16-19 and 21-34, wherein X7 is R. [Note 37] A polypeptide according to any one of the items in Notes 16 to 36, wherein X8 is Y.

[0214] [Note 38] A polypeptide according to any one of the items in Notes 16 to 36, wherein X8 is E. [Appendix 39] A polypeptide according to any one of the appendices 16 to 38, wherein X9 is D. [Appendix 40] A polypeptide according to any one of the appendices 16 to 38, wherein X9 is E.

[0215] [Note 41]X 11 A polypeptide as described in any one of the appendices 16, 17, and 21-40, wherein D is a polypeptide. [Note 42]X 11 A polypeptide as described in any one of the appendices 16, 17, and 21-40, wherein A is a polypeptide.

[0216] [Note 43]X 12 A polypeptide according to any one of the appendices 16, 17, and 21-42, wherein K is present. [Note 44]X 12 A polypeptide as described in any one of the appendices 16, 17, and 21-42, wherein A is a polypeptide.

[0217] [Note 45] X 13 A polypeptide as described in any one of the appendices 16, 17, and 21-44, wherein R is present. [Note 46]X 13A polypeptide as described in any one of the appendices 16, 17, and 21-44, wherein A is a polypeptide.

[0218] [Note 47]X 14 A polypeptide described in any one of the appendices 16 to 46, wherein R is R. [Note 48]X 14 A polypeptide described in any one of the appendices 16 to 46, wherein L is present.

[0219] [Note 49]X 15 A polypeptide as described in any one of the appendices 16-19 and 21-48, wherein F is present. [Note 50]X 15 A polypeptide according to any one of the appendices 16-19 and 21-48, wherein Y is present.

[0220] [Note 51]X 16 A polypeptide according to any one of the appendices 16-19 and 21-50, wherein K is present. [Note 52]X 16 A polypeptide as described in any one of the appendices 16-19 and 21-50, wherein R is present.

[0221] [Note 53]X 16 A polypeptide as described in any one of the appendices 16-19 and 21-50, wherein A is the polypeptide. [Note 54]X 17 A polypeptide according to any one of the appendices 16, 17, and 21-53, wherein K is present.

[0222] [Note 55]X 17 A polypeptide as described in any one of the appendices 16, 17, and 21-53, wherein A is a polypeptide. [Note 56]X 17 A polypeptide according to any one of the appendices 16, 17, and 21-53, wherein Y is present.

[0223] [Note 57]X 17 A polypeptide according to any one of the appendices 16, 17, and 21-53, wherein F is present. [Note 58]X17 A polypeptide as described in any one of the appendices 16, 17, and 21-53, wherein I is I.

[0224] [Note 59]X 18 A polypeptide described in any one of the appendices 16 to 58, wherein Q is Q. [Note 60]X 18 A polypeptide as described in any one of the appendices 16 to 58, wherein K is present.

[0225] [Note 61]X 19 A polypeptide according to any one of the appendices 16, 17, and 21-60, wherein W is present. [Note 62]X 19 A polypeptide as described in any one of the appendices 16, 17, and 21-60, wherein A is a polypeptide.

[0226] [Note 63]X 20 A polypeptide as described in any one of the appendices 16, 17, and 21-62, wherein L is present. [Note 64]X 20 A polypeptide as described in any one of the appendices 16, 17, and 21-62, wherein A is a polypeptide.

[0227] [Note 65]X 21 A polypeptide as described in any one of the appendices 16, 17, and 21-64, wherein D is a polypeptide. [Note 66]X 21 A polypeptide according to any one of the appendices 16, 17, and 21-64, wherein K is present.

[0228] [Note 67]X 21 A polypeptide as described in any one of the appendices 16, 17, and 21-64, wherein R is present. [Note 68]X 21 A polypeptide as described in any one of the appendices 16, 17, and 21-64, wherein A is a polypeptide.

[0229] [Note 69]X 21 A polypeptide according to any one of the appendices 16, 17, and 21-64, wherein F is present. [Note 70]X 21 A polypeptide as described in any one of the appendices 16, 17, and 21-64, wherein G is G.

[0230] [Note 71]X 21 A polypeptide as described in any one of the appendices 16, 17, and 21-64, wherein M is present. [Note 72]X 21 A polypeptide according to any one of the appendices 16, 17, and 21-64, wherein N is present.

[0231] [Note 73]X 21 A polypeptide as described in any one of the appendices 16, 17, and 21-64, wherein I is a polypeptide. [Note 74]X 22 A polypeptide described in any one of the appendices 16-17 and 21-73, wherein I is I.

[0232] [Note 75]X 22 A polypeptide as described in any one of the appendices 16-19 and 21-73, wherein F is present. [Note 76]X 22 A polypeptide as described in any one of the appendices 16-19 and 21-73, wherein A is a polypeptide.

[0233] [Note 77]X 23 A polypeptide as described in any one of the appendices 16 to 76, wherein K is present. [Note 78]X 23 A polypeptide as described in any one of the appendices 16 to 76, wherein T is present.

[0234] [Note 79]X 24 A polypeptide according to any one of the appendices 16, 17, 18, and 21-78, wherein K is present. [Note 80]X 24 A polypeptide as described in any one of the appendices 16, 17, 18, and 21-78, wherein E is present.

[0235] [Note 81]X 25 A polypeptide described in any one of the appendices 16 to 80, wherein D is the polypeptide. [Note 82]X 25 A polypeptide as described in any one of the appendices 16 to 80, wherein E is present.

[0236] [Note 83]X 26 A polypeptide as described in any one of the appendices 16 to 82, wherein S is present. [Note 84]X 26 A polypeptide as described in any one of the appendices 16 to 82, wherein N is present.

[0237] [Note 85]X 27 A polypeptide as described in any one of the appendices 16 to 84, wherein E is present. [Note 86]X 27 A polypeptide described in any one of the appendices 16 to 84, wherein Q is Q.

[0238] [Note 87]X 23 T is X 24 E is X 25 E is and X 26 A polypeptide as described in any one of the appendices 16 to 86, wherein N is present. [Note 88]X 23 T is X 24 K is X 25 E is and X 26 A polypeptide as described in any one of the appendices 16 to 86, wherein N is present.

[0239] [Note 89]X 17 A polypeptide as described in any one of the appendices 16 to 88, wherein K is present. [Note 90] The polypeptide according to Note 16, wherein the mutant has one of the sequences from SEQ ID NOs. 21 to 87.

[0240] [Note 91] Position X 24 A polypeptide as described in any one of the appendices 16 to 90, wherein one of the amino acids is substituted with amino acid K. [Note 92] Position X 24 A polypeptide as described in any one of the appendices 16 to 90, wherein one of the amino acids is substituted with amino acid E.

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

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

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

[0244] [Note 99] The polypeptide according to Note 98, wherein the wild-type Fc domain contains the sequence of Sequence ID No. 151. [Appendix 100] The polypeptide according to any one of Appendix 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] [Note 101] The polypeptide according to Note 100, wherein the Fc domain does not form a dimer. [Appendix 102] The polypeptide according to any one of Appendix 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 according to Appendix 102, wherein the albumin-binding peptide contains the sequence of Sequence ID No. 152. [Appendix 104] The polypeptide according to any one of the appendices 16 to 95, further comprising a fibronectin domain fused to the C-terminus of the polypeptide via a linker.

[0247] [Note 105] The polypeptide according to Note 104, wherein the fibronectin domain contains the sequence of Sequence ID No. 153. [Appendix 106] The polypeptide according to any one of the appendices 16 to 95, further comprising human serum albumin fused to the C-terminus of the polypeptide via a linker.

[0248] [Note 107] The polypeptide according to Note 106, wherein the human serum albumin contains the sequence of Sequence ID No. 154. [Note 108] A polypeptide according to Note 96 or 97 that forms a dimer.

[0249] [Appendix 109] The polypeptide according to any one of the appendices 96 to 108, wherein the linker is an amino acid spacer. [Note 110] The polypeptide according to Note 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 according to any one of the appendices 16 to 110, having a serum half-life of at least 7 days. [Note 112] K above 200 pM D A polypeptide described in any one of the appendices 16 to 111 that binds to human bone morphogenetic factor 9 (BMP9).

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

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

[0253] [Note 117] K above 5 pM D A polypeptide described in any one of the appendices 16 to 116 that binds to human GDF-11. [Note 118] A nucleic acid molecule encoding a polypeptide as described in any one of the items in Notes 16 to 117.

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

[0255] [Appendix 121] A method for producing a polypeptide as described in any one of the appendices 16 to 117, a) To provide a host cell containing a nucleic acid molecule as described in Appendix 118 or a vector as described in Appendix 119, and b) A method comprising expressing the nucleic acid molecule or vector in a host cell under conditions that enable the formation of the polypeptide.

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

[0257] [Note 123] The pharmaceutical composition according to Note 122, wherein the polypeptide is present in a therapeutically effective amount. [Appendix 124] A method for 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 Appendix 16 to 117, a nucleic acid molecule described in Appendix 118, a vector described in Appendix 119, or a pharmaceutical composition described in Appendix 122 or 123.

[0258] [Note 125] The method according to Note 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 acting on at least one signaling pathway among myostatin, activin, and BMP9 in a subject having a disease or condition accompanied by muscle weakness and muscle atrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendix 16 to 117, a nucleic acid molecule described in Appendix 118, a vector described in Appendix 119, or a pharmaceutical composition described in Appendix 122 or 123.

[0260] [Note 127] The method according to Note 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 for treating a subject having Duchenne muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendix 16 to 117, a nucleic acid molecule described in Appendix 118, a vector described in Appendix 119, or a pharmaceutical composition described in either Appendix 122 or 123.

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

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

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

[0267] [Appendix 134] A method for 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 Appendix 16 to 117, a nucleic acid molecule described in Appendix 118, a vector described in Appendix 119, or a pharmaceutical composition described in either Appendix 122 or 123.

[0268] [Appendix 135] A method for 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 Appendix 16 to 117, a nucleic acid molecule described in Appendix 118, a vector described in Appendix 119, or a pharmaceutical composition described in either Appendix 122 or 123.

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

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

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

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

[0273] [Appendix 144] The method according to any one of the Appendix 132 to 143, wherein the method reduces at least one of the amount and percentage of body fat of the subject. [Note 145] The method described above is the method described in any one of the notes 132 to 144, which does not affect the appetite for food intake of the subject.

[0274] [Appendix 146] The method according to any one of the appendices 132 to 130, wherein the method reduces the obesity of the subject. [Appendix 147] The method described above is the method described in any one of the Appendix 132 to 146, which reduces the weight of the epididymis and perirenal fat body of the subject.

[0275] [Appendix 148] The method according to any one of the Appendix 132 to 147, wherein the method reduces the amount of at least one of the subcutaneous fat and visceral fat of the subject. [Appendix 149] The method described above is the method described in any one of the Appendix 132 to 148, which reduces the fasting insulin level of the subject.

[0276] [Appendix 150] The method described above is the method described in any one of the appendices 132 to 149, which lowers the blood glucose level of the subject. [Appendix 151] The method described above is the method described in any one of the appendices 132 to 150, which enhances the insulin sensitivity of the subject.

[0277] [Appendix 152] The method described above is the method according to any one of the appendices 132 to 151, which increases the glucose clearance rate of the target. [Appendix 153] The method described above is the method described in any one of the Appendix 132 to 152, which improves the serum lipid profile of the subject.

[0278] [Appendix 154] The above method is the method described in any one of the appendices 132 to 153, which does not reduce the amount of lean body mass. [Appendix 155] The method described above is the method described in any one of the Appendix 124 to 154, which increases muscle mass.

[0279] [Appendix 156] The method according to any one of Appendix 124 to 155, wherein the method reduces or inhibits the binding of at least one of activin and myostatin to the corresponding receptor.

[0280] [Appendix 157] The method according to any one of Appendices 124-131 and 155-156, wherein a 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 act on at least one of myostatin, activin, and BMP9 in a subject, or to reduce or inhibit the binding of at least one of activin and myostatin to the corresponding receptor.

[0281] [Note 158] The method according to any one of Notes 132 to 156, wherein a 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 bodies, reduce body fat percentage, reduce body weight, reduce the rate of weight gain, lower fasting insulin levels, lower blood glucose levels, increase insulin sensitivity, act on the signaling of at least one of myostatin, activin, and BMP9 in a subject, reduce adipocyte proliferation, reduce or inhibit the binding of at least one of activin and myostatin to their corresponding receptors, reduce LDL, reduce triglycerides, improve serum lipid profile, regulate at least one of insulin biosynthesis and secretion from β-cells, delay, postpone or reduce the need for insulin, or increase glucose clearance.

[0282] [Note 159] The method described above does not cause vascular complications in the subject, and is the method described in any one of the notes 124 to 158. [Note 160] The method according to Note 159, wherein the method does not increase vascular permeability or leakage.

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

Claims

1. Includes extracellular activin type IIa receptor (ActRIIa) variant, The aforementioned variant is _|||||__________________________________________________ １ ︸ ２ !! ３ ︸ ４ ︸ ５ ︸ ６ 4???. ７ ︃ ８ ︹ ９ ︸ １０ ︸ １１ ︸ １２ ︸ １３ ︸ １４ . １５ 1) １６ *++IEQB️ １７ ︸ １８ ︣ １９ ︸ ２０ ︸ ２１ ︤ ２２ !! ff ２３ ︸ ２４ ︸ ２５ ︸ ２６ ︃ ２７ [|..|.|..|..|..|....?. It has an array of, In the array, X １ is F or Y; X ２ is F or Y; X ３ is E or A; X ４ is K or L; X ５ is D or E; X ６ is R or A; X ７ is P or R; X ８ is Y or E; X ９ is D or E; X １０ is K or Q; X １１ is 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 aforementioned mutant has at least one amino acid substitution in a wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or in an extracellular ActRIIa having one of the sequences of SEQ ID NOs: 76 to 96. Polypeptide.

2. The aforementioned mutant is GAILGRSETQECLFX ２ !! ３ ︸ ４ ︸ ５ ︸ ６ 4???. ７ ︃ ８ ︹ ９ ︹ １１ ︸ １２ ︸ １３ ︸ １４ . １５ 1) １６ *++IEQB️ １７ ︸ １８ ︣ １９ ︸ ２０ ︸ ２１ ︤ ２２ !! ff ２３ ︸ ２４ ︸ ２５ ︸ ２６ ︃ ２７ [|..|..|.|..|......?. The polypeptide according to claim 1, having the sequence of the following.

3. The aforementioned mutant is GAILGRETQECLF X ２ NANWEX ４ X ５ RTNQTGVEX ７ CX ８ GX ９ KDKRX １４ HCX １５ ATWX １６ NISGSIEIVKX １８ GCWLDDX ２２ NCYDRTDECVEX ２３ X ２４ X ２５ X ２６ PX ２７ VYFCCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3) A polypeptide according to claim 1 or 2, having the sequence of the following.

4. The aforementioned mutant is GAILGRETQECLF X ２ NANWEX ４ DRTNQTGVEX ７ CX ８ GX ９ KDKRX １４ HCX １５ ATWX １６ NISGSIEIVKX １８ GCWLDDX ２２ NCYDRTDECVEX ２３ KX ２５ X ２６ PX ２７ VYFCCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4) A polypeptide according to any one of claims 1 to 3, having the sequence of the following:

5. The aforementioned mutant is GAILGRSETQECLFX ２ NANWEX ４ DRTNQTGVEPCX ８ GX ９ KDKRX １４ HCFATWKNISGSIEIVKX １８ GCWLDDINCYDRTDCVEX ２３ KX ２５ X ２６ PX ２７ VYFCCCCEGNMCNEKFSYFPEMEVTQPTS (Sequence ID 5) A polypeptide according to any one of claims 1 to 4, having the sequence of the following:

6. X １ The polypeptide according to claim 1, wherein F is

7. X １ The polypeptide according to claim 1, wherein Y.

8. X １０ The polypeptide according to claim 1, 6, or 7, wherein is K.

9. X １０ The polypeptide according to claim 1, 6, or 7, wherein Q is Q.

10. X ２ A polypeptide according to any one of claims 1 to 9, wherein F is

11. X ２ A polypeptide according to any one of claims 1 to 9, wherein is or Y.

12. X ３ The polypeptide according to any one of claims 1, 2, and 6 to 11, wherein E is present.

13. X ３ A polypeptide according to any one of claims 1, 2, and 6 to 11, wherein A.

14. X ４ A polypeptide according to any one of claims 1 to 13, wherein is K.

15. X ４ A polypeptide according to any one of claims 1 to 13, wherein L is present.

16. X ５ A polypeptide according to any one of claims 1, 2, 3, and 6 to 15, wherein D is

17. X ５ A polypeptide according to any one of claims 1, 2, 3, and 6 to 15, wherein E is present.

18. X ６ The polypeptide according to any one of claims 1, 2, and 6 to 17, wherein R is the polypeptide.

19. X ６ A polypeptide according to any one of claims 1, 2, and 6 to 17.

20. X ７ A polypeptide according to any one of claims 1 to 4 and 6 to 19, wherein P is present.

21. X ７ A polypeptide according to any one of claims 1 to 4 and 6 to 19, wherein R is present.

22. X ８ A polypeptide according to any one of claims 1 to 21, wherein Y.

23. X ８ A polypeptide according to any one of claims 1 to 21, wherein E is

24. X ９ A polypeptide according to any one of claims 1 to 23, wherein is D.

25. X ９ A polypeptide according to any one of claims 1 to 23, wherein E is

26. X １１ A polypeptide according to any one of claims 1, 2, and 6 to 25, wherein is D.

27. X １１ A polypeptide according to any one of claims 1, 2, and 6 to 25.

28. X １２ The polypeptide according to any one of claims 1, 2, and 6 to 27, wherein is K.

29. X １２ A polypeptide according to any one of claims 1, 2, and 6 to 27.

30. X １３ A polypeptide according to any one of claims 1, 2, and 6 to 29, wherein R is present.

31. X １３ A polypeptide according to any one of claims 1, 2, and 6 to 29.

32. X １４ A polypeptide according to any one of claims 1 to 31, wherein R is present.

33. X １４ A polypeptide according to any one of claims 1 to 31, wherein L is present.

34. X １５ A polypeptide according to any one of claims 1 to 4 and 6 to 33, wherein F.

35. X １５ A polypeptide according to any one of claims 1 to 4 and 6 to 33, wherein Y.

36. X １６ The polypeptide according to any one of claims 1 to 4 and 6 to 35, wherein is K.

37. X １６ A polypeptide according to any one of claims 1 to 4 and 6 to 35, wherein R is

38. X １６ A polypeptide according to any one of claims 1 to 4 and 6 to 35, wherein A.

39. X １７ The polypeptide according to any one of claims 1, 2, and 6 to 38, wherein is K.

40. X １７ A polypeptide according to any one of claims 1, 2, and 6 to 38.

41. X １７ A polypeptide according to any one of claims 1, 2, and 6 to 38, wherein Y.

42. X １７ A polypeptide according to any one of claims 1, 2, and 6 to 38, wherein F.

43. X １７ A polypeptide according to any one of claims 1, 2, and 6 to 38, wherein is I.

44. X １８ A polypeptide according to any one of claims 1 to 43, wherein Q is Q.

45. X １８ A polypeptide according to any one of claims 1 to 43, wherein is K.

46. X １９ The polypeptide according to any one of claims 1, 2 and 6 to 45, wherein is W.

47. X １９ A polypeptide according to any one of claims 1, 2, and 6 to 45, wherein A.

48. X ２０ The polypeptide according to any one of claims 1, 2 and 6 to 47, wherein L is present.

49. X ２０ A polypeptide according to any one of claims 1, 2, and 6 to 47.

50. X ２１ The polypeptide according to any one of claims 1, 2, and 6 to 49, wherein is D.

51. X ２１ The polypeptide according to any one of claims 1, 2, and 6 to 49, wherein is K.

52. X ２１ The polypeptide according to any one of claims 1, 2, and 6 to 49, wherein R is present.

53. X ２１ A polypeptide according to any one of claims 1, 2, and 6 to 49, wherein A.

54. X ２１ A polypeptide according to any one of claims 1, 2, and 6 to 49, wherein F.

55. X ２１ The polypeptide according to any one of claims 1, 2 and 6 to 49, wherein is G.

56. X ２１ A polypeptide according to any one of claims 1, 2, and 6 to 49, wherein M is

57. X ２１ The polypeptide according to any one of claims 1, 2, and 6 to 49, wherein is N.

58. X ２１ The polypeptide according to any one of claims 1, 2 and 6 to 49, wherein is I.

59. X ２２ A polypeptide according to any one of claims 1 to 4 and 6 to 58, wherein is I.

60. X ２２ A polypeptide according to any one of claims 1 to 4 and 6 to 58, wherein F.

61. X ２２ A polypeptide according to any one of claims 1 to 4 and 6 to 58, wherein A.

62. X ２３ A polypeptide according to any one of claims 1 to 61, wherein is K.

63. X ２３ A polypeptide according to any one of claims 1 to 61, wherein T is

64. X ２４ A polypeptide according to any one of claims 1, 2, 3, and 6 to 63, wherein is K.

65. X ２４ A polypeptide according to any one of claims 1, 2, 3, and 6 to 63, wherein E is present.

66. X ２５ A polypeptide according to any one of claims 1 to 65, wherein is D.

67. X ２５ A polypeptide according to any one of claims 1 to 65, wherein E is

68. X ２６ A polypeptide according to any one of claims 1 to 67, wherein S is

69. X ２６ A polypeptide according to any one of claims 1 to 67, wherein N is

70. X ２７ A polypeptide according to any one of claims 1 to 69, wherein E is

71. X ２７ A polypeptide according to any one of claims 1 to 69, wherein Q is Q.

72. X ２３ T is X ２４ E is X ２５ E is and X ２６ A polypeptide according to any one of claims 1 to 71, wherein N is

73. X ２３ T is X ２４ K is X ２５ E is and X ２６ A polypeptide according to any one of claims 1 to 71, wherein N is

74. X １７ A polypeptide according to any one of claims 1 to 73, wherein is K.

75. The polypeptide according to claim 1, wherein the mutant has one sequence from sequence numbers 6 to 72.

76. position ２４ The polypeptide according to any one of claims 1 to 75, wherein the amino acid is substituted with amino acid K.

77. position ２４ The polypeptide according to any one of claims 1 to 75, wherein the amino acid is substituted with amino acid E.

78. The polypeptide according to any one of claims 1 to 77, further comprising a C-terminal extension of one or more amino acids.

79. The polypeptide according to claim 78, wherein the C-terminal extension is an NP.

80. The polypeptide according to claim 78, wherein the C-terminal extension is NPVTPK.

81. The polypeptide according to any one of claims 1 to 80, further comprising an Fc domain monomer fused to the C-terminus of the polypeptide via a linker.

82. The polypeptide according to claim 81, wherein the Fc domain monomer contains the sequence of sequence number 97.

83. The polypeptide according to any one of claims 1 to 80, further comprising a wild-type Fc domain fused to the C-terminus of the polypeptide via a linker.

84. The polypeptide according to claim 83, wherein the wild-type Fc domain comprises the sequence of sequence number 151.

85. The polypeptide according to any one of claims 1 to 80, further comprising an Fc domain having an amino acid substitution, fused to the C-terminus of the polypeptide via a linker.

86. The polypeptide according to claim 85, wherein the Fc domain does not form a dimer.

87. The polypeptide according to any one of claims 1 to 80, further comprising an albumin-binding peptide fused to the C-terminus of the polypeptide via a linker.

88. The polypeptide according to claim 87, wherein the albumin-binding peptide comprises the sequence of SEQ ID NO:

152.

89. The polypeptide according to any one of claims 1 to 80, further comprising a fibronectin domain fused to the C-terminus of the polypeptide via a linker.

90. The polypeptide according to claim 89, wherein the fibronectin domain comprises the sequence of Sequence ID No.

153.

91. The polypeptide according to any one of claims 1 to 80, further comprising human serum albumin fused to the C-terminus of the polypeptide via a linker.

92. The polypeptide according to claim 91, wherein the human serum albumin comprises the sequence of SEQ ID NO:

154.

93. A polypeptide according to claim 81 or 82, which forms a dimer.

94. The polypeptide according to any one of claims 81 to 93, wherein the linker is an amino acid spacer.

95. The polypeptide according to claim 94, 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).

96. A polypeptide according to any one of claims 1 to 95, having a serum half-life of at least 7 days.

97. K above 200 pM Ｄ The polypeptide according to any one of claims 1 to 96, which binds to human bone morphogenetic factor 9 (BMP9).

98. The polypeptide according to claim 97, which binds to at least one of activin and myostatin, and has low or weak binding to human BMP9.

99. The polypeptide according to claim 97 or 98, which does not substantially bind to human BMP9.

100. K below 800 pM Ｄ A polypeptide according to any one of claims 1 to 99, which binds to human activin A.

101. K below 800 pM Ｄ The polypeptide according to any one of claims 1 to 100, which binds to human activin B.

102. K above 5 pm Ｄ A polypeptide according to any one of claims 1 to 101, which binds to human GDF-11.

103. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 102.

104. A vector comprising the nucleic acid molecule of claim 103.

105. A host cell expressing a polypeptide according to any one of claims 1 to 102, comprising the nucleic acid molecule according to claim 103 or the vector according to claim 104, wherein the nucleic acid molecule or vector is expressed in the host cell.

106. A method for producing a polypeptide according to any one of claims 1 to 102, a) To provide a host cell containing the nucleic acid molecule described in claim 103 or the vector described in claim 104, and b) Expressing the nucleic acid molecule or vector in a host cell under conditions that enable the formation of the polypeptide. Methods that include...

107. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, or a vector according to claim 104 and one or more pharmaceutically acceptable carriers or excipients.

108. The pharmaceutical composition according to claim 107, wherein the polypeptide is present in a therapeutically effective amount.

109. A method for increasing muscle mass in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to claim 107 or 108.

110. The method according to claim 109, wherein the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

111. A method for acting on at least one signaling pathway among myostatin, activin, and BMP9 in a subject having a disease or condition accompanied by muscle weakness and muscle atrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to claim 107 or 108.

112. The method according to claim 111, wherein the disease or condition is Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

113. A method for treating a subject having Duchenne muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

114. A method for treating a subject having facial cystic brachial muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

115. A method for treating a subject having inclusion body myositis, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

116. A method for treating a subject having amyotrophic lateral sclerosis, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

117. A method for reducing body fat in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

118. A method for reducing body weight in a subject requiring such reduction, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

119. A method for lowering blood glucose in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

120. A method for increasing insulin sensitivity in a subject requiring it, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

121. The method according to any one of claims 117 to 120, wherein the subject has a metabolic disease or is at risk of developing such a metabolic disease.

122. A method for acting on at least one signaling pathway among myostatin, activin, and BMP9 in a subject having or at risk of developing a metabolic disorder, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

123. A method for treating and preventing at least one of metabolic diseases in a subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 102, a nucleic acid molecule according to claim 103, a vector according to claim 104, or a pharmaceutical composition according to any one of claims 107 or 108.

124. The method according to any one of claims 121 to 123, wherein the metabolic disease is selected from the group consisting of obesity, type 1 diabetes and type 2 diabetes.

125. The method according to claim 124, wherein the metabolic disorder is obesity.

126. The method according to claim 124, wherein the metabolic disease is type 1 diabetes.

127. The method according to claim 124, wherein the metabolic disease is type 2 diabetes.

128. The method according to any one of claims 117 to 127, wherein the method reduces at least one of the target's weight and the rate of weight gain.

129. The method according to any one of claims 117 to 128, wherein the method reduces at least one of the amount of body fat and the percentage of body fat of the subject.

130. The method according to any one of claims 117 to 129, wherein the method does not affect the appetite for food intake of the subject.

131. The method according to any one of claims 117 to 130, wherein the method reduces obesity in the subject.

132. The method according to any one of claims 117 to 131, wherein the method reduces the weight of the epididymis and perirenal fat body of the subject.

133. The method according to any one of claims 117 to 132, wherein the method reduces the amount of at least one of the subcutaneous fat and visceral fat of the target.

134. The method according to any one of claims 117 to 133, wherein the method reduces the fasting insulin level of the subject.

135. The method according to any one of claims 117 to 134, wherein the method lowers the blood glucose level of the target.

136. The method according to any one of claims 117 to 135, wherein the method enhances the insulin sensitivity of the subject.

137. The method according to any one of claims 117 to 136, wherein the method increases the glucose clearance rate of the target.

138. The method according to any one of claims 117 to 137, wherein the method improves the serum lipid profile of the subject.

139. The method according to any one of claims 117 to 138, wherein the method does not reduce the amount of lean body mass.

140. The method according to any one of claims 109 to 139, wherein the method increases muscle mass.

141. The method according to any one of claims 109 to 140, wherein the method reduces or inhibits the binding of at least one of activin and myostatin to the corresponding receptor.

142. The method according to any one of claims 109-116 and 140-141, wherein a 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 act on at least one of myostatin, activin, and BMP9 in a subject, or to reduce or inhibit the binding of at least one of activin and myostatin to the corresponding receptor.

143. The method according to any one of claims 117 to 141, wherein a 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 bodies, reduce body fat percentage, reduce body weight, reduce the rate of weight gain, lower fasting insulin levels, lower blood glucose levels, increase insulin sensitivity, act on the signaling of at least one of myostatin, activin, and BMP9 in a subject, reduce adipocyte proliferation, reduce or inhibit the binding of at least one of activin and myostatin to their corresponding receptors, reduce LDL, reduce triglycerides, improve serum lipid profile, regulate at least one of insulin biosynthesis and secretion from β-cells, delay, postpone or reduce the need for insulin, or increase glucose clearance.

144. The method according to any one of claims 109 to 143, wherein the method does not cause vascular complications in the subject.

145. The method according to claim 144, wherein the method does not increase vascular permeability or leakage.

146. The method according to any one of claims 109 to 145, wherein the method increases the bone mineral density of the target.

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