Activin receptor IIA variants and methods of use thereof

Abstract

The present invention relates to activin receptor IIA type variants and methods of use thereof. The present invention features a polypeptide comprising an extracellular ActRIIa variant. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIa variant fused to an Fc domain monomer or part. The invention also features the use of said polypeptides for the treatment of diseases and conditions involving bone injury, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer or cancer metastasis-associated bone loss, Paget's disease, renal bone dystrophy, treatment-associated bone loss, diet-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, osteoporosis-associated bone loss, and osteoporosis-associated bone loss. Pharmaceutical compositions and methods for treating bone loss associated with obesity treatment, low gravity associated bone loss, or immobility associated bone loss.

Description

[0001] This application is a divisional application of the invention patent application entitled “Activin receptor type IIA variants and methods of use thereof” which entered into China with application number 201780082824.X and international application date PCT / US2017 / 060970 on November 9, 2017.

[0002] Technical Field and Background Art Healthy bones undergo constant remodeling involving bone destruction and bone growth. Bone growth is mediated by osteoblastic cell types, while osteoclasts resorb bone. Pathology occurs when these systems lose balance through downregulation of anabolic programs, upregulation of catabolic systems, or a combination of both, resulting in net bone loss. Therefore, controlling the balance in bone remodeling can be used to promote fracture healing and other injuries to the skeleton, as well as to treat conditions associated with bone loss and bone mineralization, such as osteoporosis.

[0003] Bone damage can be caused by a series of underlying causes (including age- or cancer-related bone loss, genetic conditions, adverse side effects of drug treatments, or fractures). The World Health Organization estimates that osteoporosis affects 75 million people in the United States, Europe, and Japan alone, and is an important risk factor for fractures. Typically, overall bone loss represents a pathological state with few effective treatments. Instead, treatment focuses on immobilization, exercise, and dietary adjustments, rather than agents that directly promote bone growth and increase bone density. Regarding osteoporosis, estrogen, calcitonin, osteocalcin, and vitamin K or high-dose dietary calcium are used as therapeutic interventions. Other treatments for osteoporosis include bisphosphonates, parathyroid hormone, parathyroid hormone-related protein (PTHrP) calcimimetic agents, statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. However, such therapeutic agents are often associated with undesirable side effects. For diseases that cause bone damage or bone demineralization, new and effective treatments are needed. Summary of the invention The present invention features a polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant. In some embodiments, the polypeptide of the present invention comprises an extracellular ActRIIa variant fused to the N- or C-terminus of an Fc domain monomer or portion. Such portions may be linked by amino acids or other covalent bonds. Polypeptides comprising extracellular ActRIIa variants fused to Fc domain monomers may also form dimers (e.g., homodimers or heterodimers) by interactions between two Fc domain monomers. The polypeptides of the present invention can be used to increase bone mass or bone mineral density in a subject having a disease or condition involving bone damage, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss or immobility. Further, the polypeptides of the present invention can also be used to affect myostatin, activin and / or bone morphogenetic protein 9 (BMP9) signaling in a subject at risk of developing or having a disease or condition involving bone damage or bone demineralization.

[0005] In one aspect, the invention features a polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant having the following sequence: GAILGRSETQECLX 1 X 2 NANWX 3 X 4 X 5 X 6 TNQTGVEX 7 CX 8 GX 9 X 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 27VYFCCCEGNMCNEKF SYFPEMEVTQPTS (SEQ ID NO: 1), wherein 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 It is K or Q; X 11 It is D or A; X 12 It is K or A; X 13 is R or A; X 14 is R or L; X 15 is F or Y; X 16 is K, R or A; X 17 is K, A, Y, F or I; X 18 Q or K; X 19 W or A; X 20 L or A; X 21 is D, K, R, A, F, G, M, N, or I; X 22 is I, F or A; X 23 is K or T; X 24 Is K or E; X 25 Is D or E; X 26 is S or N; and X 27 is E or Q, and wherein the variant has at least one amino acid substitution relative to wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or an extracellular ActRIIa having any one of the sequences of SEQ ID NOs: 76-96.

[0006] In some embodiments, the variant has the following sequence: GAILGRSETQECLFX 2 NANWX 3 X 4 X 5 X 6 TNQTGVEX 7 CX 8 GX 9 KX 11 X 12 X 13 X 14 HCX 15 ATWX 16 NI SGSIEIVX 17 X 18 GCX19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSY FPEMEVTQPTS (SEQ ID NO: 2), wherein X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 As defined above.

[0007] In some embodiments, the variant has the following sequence: GAILGRSETQECLFX 2 NANWEX 4 X 5 RTNQTGVEX 7 CX 8 GX 9 KDKRX 14 HCX 15 ATWX 16 NISGSIE IVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQ PTS (SEQ ID NO: 3), wherein X 2 , X 4 , X 5 , X7 , X 8 , X 9 , X 14 , X 15 , X 16 , X 18 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 As defined above.

[0008] In some embodiments, the variant has the following sequence: GAILGRSETQECLFX 2 NANWEX 4 DRTNQTGVEX 7 CX 8 GX 9 KDKRX 14 HCX 15 ATWX 16 NISGSIEI VKX 18 GCWLDDX 22 NCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4), wherein X 2 , X 4 , X 7 , X 8 , X 9 , X 14 , X 15 , X 16 , X 18 , X 22 , X 23 , X 25 , X 26 and X 27 As defined above.

[0009] In some embodiments, the variant has the following sequence: GAILGRSETQECLFX 2 NANWEX 4 DRTNQTGVEPCX 8 GX 9 KDKRX 14 HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5), wherein X 2 , X 4 , X 8 , X 9 , X 14 , X 18 , X 23 , X 25 , X 26 and X 27 As defined above.

[0010] In any of the preceding embodiments, X 1 is F or Y. In any of the preceding embodiments, X 2 is F or Y. In any of the preceding embodiments, X 3 is E or A. In any of the preceding embodiments, X 4 is K or L. In any of the preceding embodiments, X 5 is D or E. In any of the preceding embodiments, X 6 is R or A. In any of the foregoing embodiments, X 7 is P or R. In any of the preceding embodiments, X 8 is Y or E. In any of the preceding embodiments, X 9 is D or E. In any of the preceding embodiments, X 10 is K or Q. In any of the foregoing embodiments, X 11 is D or A. In any of the foregoing embodiments, X 12 is K or A. In any of the preceding embodiments, X 13 is R or A. In any of the foregoing embodiments, X 14 is R or L. In any of the preceding embodiments, X 15 is F or Y. In any of the preceding embodiments, X 16 is K, R or A. In any of the foregoing embodiments, X 17 is K, A, Y, F or I. In any of the foregoing embodiments, X 18 is Q or K. In any of the foregoing embodiments, X 19 is W or A. In any of the preceding embodiments, X 20 is L or A. In any of the preceding embodiments, X 21 is D, K, R, A, F, G, M, N or I. In any of the foregoing embodiments, X 22 is I, F or A. In any of the foregoing embodiments, X 23is K or T. In any of the preceding embodiments, X 24 is K or E. In any of the preceding embodiments, X 25 is D or E. In any of the preceding embodiments, X 26 is S or N. In any of the foregoing embodiments, X 27 is E or Q. In any of the foregoing embodiments, X 23 It's T, X 24 Yes E, X 25 is E, and X 26 It is N, or X 23 It's T, X 24 It is K, X 25 is E, and X 26 is N. In any of the preceding embodiments, X 17 It's K.

[0011] In any of the preceding embodiments, the variant has the sequence of any one of SEQ ID NOs: 6-72.

[0012] In any of the preceding embodiments, position X 24 The amino acid at the position can be replaced by amino acid K.

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

[0014] In any of the foregoing embodiments, the polypeptides described herein may further comprise a C-terminal extension of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids). In some embodiments, the C-terminal extension is the amino acid sequence NP. In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 155).

[0015] In any of the foregoing embodiments, the polypeptides described herein may further comprise a moiety fused or covalently linked to the C-terminus of the polypeptide. The moiety may increase the stability of the polypeptide or improve the pharmacokinetic properties of the polypeptide. In some embodiments, the moiety is an Fc domain monomer, an Fc domain, an albumin binding peptide, a fibronectin domain, or serum albumin.

[0016] In any of the foregoing embodiments, the polypeptide described herein may further comprise an Fc domain monomer fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant described herein fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers. In some embodiments, the Fc domain monomer has a sequence of SEQ ID NO: 97.

[0017] In any of the foregoing embodiments, the polypeptide described herein may further comprise an Fc domain fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the Fc domain is a wild-type Fc domain. In some embodiments, the wild-type Fc domain has a sequence of SEQ ID NO: 151. In some embodiments, the Fc domain contains one or more amino acid substitutions. In some embodiments, the Fc domain containing one or more amino acid substitutions does not form a dimer.

[0018] In any of the foregoing embodiments, the polypeptide described herein may further comprise an albumin binding peptide fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the albumin binding peptide has the sequence of SEQ ID NO:152.

[0019] In any of the foregoing embodiments, the polypeptide described herein may further comprise a fibronectin domain fused to the C-terminus of the polypeptide by means of a linker.In some embodiments, the fibronectin domain peptide has the sequence of SEQ ID NO:153.

[0020] In any of the foregoing embodiments, the polypeptide described herein may further comprise human serum albumin fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the human serum albumin has a sequence of SEQ ID NO: 154.

[0021] 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).

[0022] In some embodiments, the amino acid spacer is GGGS (SEQ ID NO:99), GGGGA (SEQ ID NO:101), GGGGS (SEQ ID NO:102), GGGGG (SEQ ID NO:103), GGAG (SEQ ID NO:104), GGSG (SEQ ID NO:105), AGGG (SEQ ID NO:106), SGGG (SEQ ID NO:107), GAGA (SEQ ID NO:108), GSGS (SEQ ID NO:109), GAGAGA (SEQ ID NO:110), GSGSGS (SEQ ID NO:111), GAGAGAGA (SEQ ID NO:112), GSGSGSGS (SEQ ID NO:113), GAGAGAGAGA (SEQ ID NO:114), GSGSGSGSGS (SEQ ID NO:115), GAGAGAGAGAGA (SEQ ID NO:116), and GSGSGSGSGSGS (SEQ ID NO:117), GGAGGA (SEQ ID NO:118), GGSGGS (SEQ ID NO:119), GGAGGAGGA (SEQ ID NO:120), GGSGGSGGS (SEQ ID NO:121), GGAGGAGGAGGA (SEQ ID NO:122), GGSGGSGGSGGS (SEQ ID NO:123), GGAGGGAG (SEQ ID NO:124), GGSGGGSG (SEQ ID NO:125), GGAGGGAGGGAG (SEQ ID NO:126), and GGSGGGSGGGSG (SEQ ID NO:127), GGGGGAGGGGAGGGGA (SEQ ID NO:128), GGGGSGGGGSGGGGS (SEQ ID NO:129), AAAL (SEQ ID NO:133), AAAK (SEQ ID NO:134), AAAR (SEQ ID NO:135), EGKSSGSGSESKST (SEQ ID NO:136), GSAGSAAGSGEF (SEQ ID NO:137), AEAAAKEAAAKA (SEQ ID NO:138), KESGSVSSEQLAQFRSLD (SEQ ID NO:139), GENLYFQSGG (SEQ ID NO:140), SACYCELS (SEQ ID NO:141), RSIAT (SEQ IDNO:142), RPACKIPNDLKQKVMNH (SEQ ID NO:143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:144), AAANSSIDLISVPVDSR (SEQ ID NO:145), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:146), EAAAK (SEQ ID NO:146) NO:147) or PAPAP (SEQ ID NO:148).

[0023] In any of the preceding embodiments, the polypeptide described herein has a serum half-life of at least 7 days.

[0024] In any of the preceding embodiments, the polypeptides described herein have a K of 200 pM or more. D Binds to human bone morphogenetic protein 9 (BMP9). In some embodiments, the polypeptide binds to activin and / or myostatin and has reduced (eg, weak) binding to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0025] In any of the preceding embodiments, the polypeptides described herein have a K of 800 pM or less. D Binds human activin A.

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

[0027] In any of the preceding embodiments, the polypeptides described herein have a K of approximately 5 pM or higher. D Binds human GDF-11.

[0028] In another aspect, the invention features a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72). In another aspect, the invention also features a vector comprising a nucleic acid molecule described herein.

[0029] In another aspect, the invention features a host cell expressing a polypeptide described herein, wherein the host cell comprises a nucleic acid molecule or a vector described in the first two aspects, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0030] In another aspect, the invention features a method of making a polypeptide described herein, wherein the method includes: a) providing a host cell comprising a nucleic acid molecule or a vector described herein, and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow formation of the polypeptide.

[0031] In another aspect, the invention features a pharmaceutical composition comprising a polypeptide, nucleic acid molecule or vector as described herein and one or more pharmaceutically acceptable carriers or excipients. In some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule or vector is in a therapeutically effective amount.

[0032] In another aspect, the invention also features a construct (e.g., a homodimer) comprising two identical polypeptides, each comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72) fused to the N- or C-terminus of an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0033] In another aspect, the invention also features a construct (e.g., a heterodimer) comprising two different polypeptides, each comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72) fused to the N- or C-terminus of an Fc domain monomer. The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0034] In another aspect, the invention features a method of increasing bone mineral density in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule or vector described herein or a pharmaceutical composition described herein.

[0035] In another aspect, the invention features a method of reducing bone resorption in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule or vector described herein or a pharmaceutical composition described herein.

[0036] In another aspect, the invention features a method of increasing bone formation in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule or vector described herein or a pharmaceutical composition described herein.

[0037] In some embodiments of any of the aforementioned aspects, the patient has primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobility-related bone loss.

[0038] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to its receptor) in a subject having a disease or condition involving bone damage, wherein the method comprises administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments of this aspect, the disease or condition is primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobility-related bone loss.

[0039] In another aspect, the invention features a method of treating a subject having primary osteoporosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0040] In another aspect, the invention features a method of treating a subject having secondary osteoporosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0041] In another aspect, the invention features a method of treating a subject having osteopenia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0042] In another aspect, the invention features a method of treating a subject having a bone fracture by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0043] In another aspect, the invention features a method of treating a subject having bone cancer or cancer metastasis-associated bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0044] In another aspect, the invention features a method of treating a subject having Paget's disease by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0045] In another aspect, the invention features a method of treating a subject having renal osteodystrophy by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0046] In another aspect, the invention features a method of treating a subject having therapy-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0047] In another aspect, the invention features a method of treating a subject with diet-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0048] In another aspect, the invention features a method of treating a subject with bone loss associated with obesity treatment by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0049] In another aspect, the invention features a method of treating a subject with low gravity-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0050] In another aspect, the invention features a method of treating a subject with immobility-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0051] In some embodiments of any of the aforementioned aspects, the primary osteoporosis is age-related osteoporosis.

[0052] In some embodiments of any of the aforementioned aspects, the primary osteoporosis is hormone-related osteoporosis.

[0053] In some embodiments of any of the aforementioned aspects, the secondary osteoporosis is immobilization-induced osteoporosis.

[0054] In some embodiments of any of the aforementioned aspects, wherein the secondary osteoporosis is glucocorticoid-induced osteoporosis.

[0055] In some embodiments of any of the aforementioned aspects, the cancer is multiple myeloma.

[0056] In some embodiments of any of the aforementioned aspects, the treatment is FGF-21 treatment.

[0057] In some embodiments of any of the above aspects, the therapy is GLP-1 therapy.

[0058] In some embodiments of any of the aforementioned aspects, the treatment is cancer therapy.

[0059] In some embodiments of any of the aforementioned aspects, the treatment is treatment of obesity and / or type 2 diabetes.

[0060] In some embodiments of any of the above aspects, the diet-related bone loss is rickets.

[0061] In some embodiments of any of the above aspects, the method increases bone formation in a subject. In some embodiments of any of the above aspects, wherein the method reduces bone resorption in a subject. In some embodiments of any of the above aspects, the method increases osteoblast activity or osteoblastogenesis. In some embodiments of any of the above aspects, the method reduces osteoclast activity or reduces osteoclastogenesis. In some embodiments of any of the above aspects, the method reduces or inhibits the binding of activin and / or myostatin to its receptor.

[0062] In some embodiments of any of the methods described herein, the method does not cause vascular complications in the subject (eg, increased vascular permeability or leakage). In some embodiments of any of the methods described herein, the method increases bone mineral density in the subject.

[0063] In some embodiments of any of the above aspects, the bone is cortical bone. In some embodiments of any of the above aspects, the bone is trabecular bone.

[0064] In some embodiments of any of the aforementioned aspects, the polypeptide, nucleic acid, vector or pharmaceutical composition is administered in an amount sufficient to increase bone density, reduce bone resorption, reduce the rate of bone resorption, increase bone formation, increase the rate of bone formation, reduce osteoclast activity, increase osteoblast activity, or affect myostatin, activin and / or BMP9 signaling in a subject.

[0065] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 69. In some embodiments, the variant having a sequence of SEQ ID NO: 69 has a position X 17 The amino acid K at position X 23 , X24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 69 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0066] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 58. In some embodiments, the variant having a sequence of SEQ ID NO: 58 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 58 (optionally having position X 17 The amino acid K at position X 23 , X24 , X 25 and X 26 The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0067] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 6. In some embodiments, the variant having a sequence of SEQ ID NO: 6 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 6 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0068] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 38. In some embodiments, the variant having a sequence of SEQ ID NO: 38 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 38 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the position of the amino acid sequence TEEN or TKEN, and / or a C-terminal extension ...

[0069] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 41. In some embodiments, the variant having a sequence of SEQ ID NO: 41 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 41 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0070] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 44. In some embodiments, the variant having a sequence of SEQ ID NO: 44 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 44 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0071] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 70. In some embodiments, the variant having a sequence of SEQ ID NO: 70 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 70 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0072] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 71. In some embodiments, the variant having a sequence of SEQ ID NO: 71 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 71 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of the amino acid sequence TEEN or TKEN at ... The invention relates to a condition or disease involving bone damage in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss), or affects myostatin, activin and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity therapy, low gravity-related bone loss, or immobility-related bone loss).

[0073] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 72. In some embodiments, the variant having a sequence of SEQ ID NO: 72 has a position X 17 The amino acid K at position, and / or position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 72 (optionally having an amino acid sequence at position X) 17 The amino acid K at position and / or position X 23 , X 24 , X 25 and X 26The invention relates to a method of increasing bone mineral density, increasing bone formation, decreasing bone resorption, or treating a condition or disease involving bone damage in a subject in need thereof (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss), or affecting myostatin, activin, and / or BMP9 signaling in a subject (e.g., a subject with primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss).

[0074] definition As used herein, the term "extracellular activin receptor type IIa (ActRIIa) variant" refers to a peptide comprising a soluble, extracellular portion of a single transmembrane receptor ActRIIa having at least one amino acid substitution relative to wild-type extracellular ActRIIa (e.g., the bold portion of the sequence of SEQ ID NO: 75 shown below) or an extracellular ActRIIa having any of the sequences of SEQ ID NOs: 76-96. The sequence of the wild-type, human ActRIIa precursor protein is shown below (SEQ ID NO: 75), wherein the signal peptide is italicized and the extracellular portion is in bold.

[0075] Wild-type, human ActRIIa precursor protein (SEQ ID NO: 75):

[0076] The extracellular ActRIIa variant may have a sequence of any one of SEQ ID NOs: 1-72. In specific embodiments, the extracellular ActRIIa variant has a sequence of any one of SEQ ID NOs: 6-72 (Table 2). In some embodiments, the extracellular ActRIIa variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97% or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIa (SEQ ID NO: 73).

[0077] As used herein, the term "extracellular ActRIIb variant" refers to a peptide comprising a soluble, extracellular portion of ActRIIb of a single transmembrane receptor ActRIIb having at least one amino acid substitution relative to wild-type extracellular ActRIIb (e.g., the sequence of SEQ ID NO: 74). The extracellular ActRIIb variant may have the sequence of SEQ ID NO: 149 as shown below: Extracellular ActRIIb variant (SEQ ID NO: 149):

[0078] As used herein, the term "linker" refers to a connection between two elements (e.g., a peptide or protein domain). The polypeptide described herein may include an extracellular ActRIIa variant fused to a portion (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)). The portion may increase stability or improve the pharmacokinetic properties of the polypeptide. The portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide by means of a linker. The linker may be a covalent bond or a spacer. The term "bond" refers to a chemical bond, such as an amide bond or a disulfide bond, or any type of bond produced by a chemical reaction (e.g., chemical conjugation). The term "spacer" refers to a portion (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence) that exists between two elements (e.g., peptides or protein domains) to provide space and / or flexibility between the two elements. The amino acid spacer is part of the primary sequence of the polypeptide (e.g., fused to the spacer peptide via the polypeptide backbone). For example, the formation of a disulfide bond between the two hinge regions that form the Fc domain is not considered a linker.

[0079] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain is a polypeptide having at least C H 2 domains and C H The human Fc domain of the three domains has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97% or 100% sequence identity). The Fc domain monomer comprises the second and third antibody constant domains (C H 2 and C H 3). In some embodiments, the Fc domain monomer further comprises a hinge domain. The Fc domain does not comprise any portion of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are connected by two CH 3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. In some embodiments, the Fc domain can be mutated to lack effector function, which is a typical "death Fc domain". In certain embodiments, each Fc domain monomer in the Fc domain comprises C H 2. Amino acid substitutions in the antibody constant domain that reduce the interaction or binding between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA or IgD. In addition, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3 or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0080] As used herein, the term "albumin binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has an affinity for serum albumin and a function of binding to serum albumin. The albumin binding peptide can be of different origins (e.g., human, mouse or rat). In some embodiments, the albumin binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0081] As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix or a fragment thereof that binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: 153) having amino acids 610-702 of the sequence of UniProt ID NO: P02751. In other embodiments, the fibronectin domain is an adnectin protein.

[0082] As used herein, the term "human serum albumin" refers to albumin present in human plasma. Human serum albumin is the most abundant protein in blood. It constitutes about half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0083] As used herein, the term "fusion" is used to describe the combination or attachment of two or more elements, components or protein domains (e.g., peptides or polypeptides) by means including chemical conjugation, recombinant means and chemical bonds (e.g., amide bonds). For example, two single peptides in a tandem series can be fused to form a continuous protein structure, such as a polypeptide, by chemical conjugation, chemical bonds, peptide linkers or any other means of covalent attachment. In some embodiments of the polypeptides described herein, an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of a portion (e.g., an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., a sequence of SEQ ID NO: 151), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide (e.g., a sequence of SEQ ID NO: 152), a fibronectin domain (e.g., a sequence of SEQ ID NO: 153), or human serum albumin (e.g., a sequence of SEQ ID NO: 154)) in a tandem series by means of a linker. For example, the extracellular ActRIIa variant is fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by means of a peptide linker, wherein the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIa variant by a chemical bond (e.g., a peptide bond), and the C-terminus of the peptide linker is fused to the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by a chemical bond (e.g., a peptide bond).

[0084] As used herein, the term "C-terminal extension" refers to the addition of one or more amino acids to a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). The C-terminal extension can be one or more amino acids (e.g., 1, 2, 3, 4, 5, 6 or more amino acids). Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 155) (a six amino acid C-terminal extension). Any amino acid sequence that does not destroy the activity of the polypeptide can be used. SEQ ID NO: 71 (which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NP) and SEQ ID NO: 72 (which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NPVTPK) represent two possible ways in which a polypeptide of the invention can be modified to include a C-terminal extension.

[0085] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence (e.g., an extracellular ActRIIa variant) that are identical to amino acid (or nucleic acid) residues of a reference sequence (e.g., wild-type extracellular ActRIIa (e.g., SEQ ID NO: 73)), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and nonhomologous sequences can be ignored for comparison purposes). Alignment for the purpose of determining percent identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which may alternatively be expressed as a given candidate sequence having or comprising a particular percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows: 100 x (fraction of A / B) wherein A is the number of amino acid (or nucleic acid) residues that score as identical in the alignment of the candidate sequence and the reference sequence, and wherein B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence is not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0086] In a specific embodiment, the reference sequence for comparison with the candidate sequence can be shown to be 50% to 100% identical to the candidate sequence over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence compared for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90% or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, the molecules are identical at that position.

[0087] As used herein, the term "serum half-life" refers to the time required for the plasma concentration of a protein in a subject to be reduced by half in the context of administering a therapeutic protein to a subject. Proteins can be redistributed or cleared from the bloodstream, or, for example, degraded by proteolysis. As described herein, polypeptides comprising extracellular ActRIIa variants (e.g., extracellular ActRIIa variants having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) exhibit a serum half-life of 7 days in humans. As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Typically, binding affinity refers to the strength of the sum of the non-covalent interactions between a molecule and its binding partner (such as an extracellular ActRIIa variant and BMP9 or activin A). Unless otherwise indicated, binding affinity refers to intrinsic binding affinity, which reflects a 1: 1 interaction between members of a binding pair. The binding affinity between two molecules is generally measured by the dissociation constant (K D ) or affinity constant (K A ). Two molecules with low binding affinity for each other typically associate slowly, tend to dissociate easily, and exhibit a large K D Two molecules that have a high affinity for each other generally bind easily, tend to remain bound longer, and exhibit a small K D The K of two interacting molecules can be determined using methods and techniques well known in the art, such as surface plasmon resonance. D . K D Calculated as k off / k on ratio.

[0088] As used herein, the terms "bone mineral density (BMD)", "bone density" and "bone mass" refer to a measure of the amount of bone mineral (e.g., calcium) in bone tissue. BMD can be measured by well-established clinical techniques known to those skilled in the art (e.g., by single-1 or dual-energy photon or X-ray absorptiometry). The concept of BMD relates to the amount of minerals per volume of bone, although clinically it is measured by surrogate based on the optical density per square centimeter of bone surface during imaging. BMD measurements are used as indirect indicators of osteoporosis and fracture risk in clinical medicine. In some embodiments, the BMD test results are provided as T-scores, wherein the T-scores represent the BMD of the subject compared to the ideal or peak bone mineral density of a healthy 30-year-old adult. A score of 0 indicates that BMD is equal to the normal reference value for a healthy young adult. The difference between the measured subject's BMD and the reference value for a healthy young adult is measured in standard deviation units (SD). Thus, a T-score between +1 SD and -1 SD may indicate normal BMD, a T-score between -1 SD and -2.5 SD may indicate low bone mass (e.g., osteopenia), and a T-score below -2.5 SD may indicate osteoporosis or severe osteoporosis. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention is administered to a subject in need thereof, wherein the patient has low bone mass (e.g., a T-score between -1 SD and -2.5 SD). In some embodiments, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention is administered to a subject in need thereof, wherein the patient has osteoporosis (e.g., a T-score of less than -2.5 SD). In some embodiments, administration of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention treats the subject by increasing its BMD.In some embodiments, administration of a polypeptide of the invention comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule increases BMD in a subject, resulting in an increase in the subject's T-score (e.g., resulting in an increase in the subject's T-score by 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 1.0 or more, or 2.0 or more).

[0089] As used herein, the term "bone remodeling" or "bone metabolism" refers to the process of maintaining bone strength and ion homeostasis by replacing discrete portions of old bone with newly synthesized protein matrix packets. Bone is resorbed by osteoclasts and deposited by osteoblasts in a process called ossification. Osteocyte activity plays a key role in this process. Conditions that lead to decreased bone mass can be caused by an increase in resorption or a decrease in ossification. In healthy individuals, during childhood, bone formation exceeds resorption. As the aging process occurs, resorption exceeds formation. Bone resorption rates are also typically much higher in older women after menopause due to estrogen deficiency associated with menopause.

[0090] As used herein, the term "bone resorption" or "bone catabolic activity" refers to the process by which osteoclasts break down tissue in the bone and release minerals, resulting in the transfer of minerals (e.g., calcium) from the bone tissue to the blood. An increase in the rate of bone resorption is associated with aging, including postmenopausal women. A high bone resorption rate, or a bone resorption rate that exceeds the ossification rate, is associated with skeletal disorders such as decreased bone mineral density, including osteopenia and osteoporosis. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant of the invention (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule is administered to a subject in need thereof to reduce bone resorption in the subject (e.g., the rate of bone resorption in the subject).

[0091] As used herein, the terms "bone formation," "ossification," "osteogenesis," or "bone anabolic activity" refer to the process by which new bone tissue is formed by osteoblasts. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention is administered to a subject in need thereof to increase bone formation (e.g., increase the rate of bone formation or bone formation in a subject).

[0092] As used herein, the phrase "affecting myostatin, activin and / or BMP9 signaling" refers to altering the binding of myostatin, activin and / or BMP9 to its receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). In some embodiments, a polypeptide comprising an extracellular ActRIIa variant described herein reduces or inhibits the binding of myostatin, activin and / or BMP9 to its receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). As described herein, a polypeptide of the invention comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may have a weak binding affinity for BMP9 (e.g., a K of 200 pM or more). D ).

[0093] As used herein, the term "vascular complication" refers to a vascular disorder or any damage to a blood vessel, such as damage to a blood vessel wall. Damage to the blood vessel wall can cause an increase in vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the blood vessel wall to allow small molecules, proteins, and cells to flow into and out of the blood vessel. An increase in vascular permeability or leakage may be caused by an increase in the gaps between endothelial cells lining the blood vessel wall (e.g., an increase in the size and / or number of the gaps) and / or thinning of the blood vessel wall.

[0094] As used herein, the term "polypeptide" describes a single polymer in which monomers are amino acid residues covalently joined together by amide bonds. Polypeptide is intended to encompass any amino acid sequence occurring naturally, recombinantly, or synthetically produced.

[0095] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules (such as proteins or nucleic acids). Two identical monomers can form a homodimer by covalent or non-covalent bonds. For example, if two Fc domain monomers contain the same sequence, the Fc domain can be a homodimer of two Fc domain monomers. In another example, a polypeptide described herein comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a homodimer by the interaction of two Fc domain monomers, which form an Fc domain in a homodimer.

[0096] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules (such as proteins or nucleic acids). Two monomers can form a heterodimer by covalent or non-covalent bonds. For example, a polypeptide described herein comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a heterodimer by the interaction of two Fc domain monomers (each of which is fused to a different ActRIIa variant), wherein the Fc domain monomers form an Fc domain in the heterodimer.

[0097] As used herein, the term "host cell" refers to a medium containing the necessary cellular components (e.g., organelles) required for expressing proteins from its corresponding nucleic acid. Nucleic acids are generally included in nucleic acid vectors, which can be introduced into host cells by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells can be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells).

[0098] As used herein, the term "therapeutically effective amount" refers to the amount of a polypeptide, nucleic acid or vector of the present invention or a pharmaceutical composition containing a polypeptide, nucleic acid or vector of the present invention that is effective in achieving the desired therapeutic effect in treating a patient with a disease, such as osteoporosis, or a condition involving bone damage, for example, primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss. The term "therapeutically effective amount" also refers to the amount of a polypeptide, nucleic acid or vector of the present invention or a pharmaceutical composition containing a polypeptide, nucleic acid or vector of the present invention that is effective in achieving the desired therapeutic effect in treating a patient with a disease, such as a disease or condition involving bone damage. Specifically, a therapeutically effective amount of a polypeptide, nucleic acid or vector avoids adverse side effects.

[0099] As used herein, the term "pharmaceutical composition" refers to a medicine or pharmaceutical preparation comprising an active ingredient and excipients and diluents that enable the active ingredient to be suitable for the method of administration. The pharmaceutical composition of the present invention comprises pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid or vector. The pharmaceutical composition can be in the form of tablets or capsules for oral administration, or in an aqueous form for intravenous or subcutaneous administration.

[0100] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not harmful to the recipient. In the present invention, a pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutical stability for a polypeptide comprising an extracellular ActRIIa variant, a nucleic acid molecule encoding the polypeptide, or a vector containing such a nucleic acid molecule. The nature of the carrier or excipient varies with the mode of administration. For example, for intravenous administration, an aqueous carrier is generally used; for oral administration, a solid carrier is preferred.

[0101] As used herein, the term "treating and / or preventing" refers to the use of the methods and compositions of the present invention to treat and / or prevent a disease, such as a bone disease or condition (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss). Typically, treating a bone disease or condition occurs after a subject has developed a bone disease or condition and / or has been diagnosed with a bone disease or condition. Preventing a bone disease or condition refers to steps or procedures taken when a subject is at risk of developing a bone disease or condition. A subject may show signs or mild symptoms that are judged by a physician to be signs or risk factors for developing a bone disease or condition, or have a family history or genetic predisposition to developing a bone disease or condition, but has not yet developed the disease.

[0102] As used herein, the term "subject" refers to a mammal, such as preferably a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., cynomolgus monkeys), mice, dogs, cats, horses, and cows, etc. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIa and ActRIIb and the amino acid substitutions in the ActRIIa variants. DETAILED DESCRIPTION The invention features polypeptides comprising extracellular activin receptor type IIa (ActRIIa) variants. In some embodiments, the polypeptides of the invention comprise an extracellular ActRIIa variant fused to a portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIa variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The ActRIIa variants described herein have weak or no binding affinity for bone morphogenetic protein 9 (BMP9) compared to activin and myostatin. The invention also includes methods of treating diseases and conditions involving bone damage by increasing bone mineral density or bone formation or affecting myostatin, activin and / or BMP9 signaling in a subject by administering to the subject a polypeptide comprising an extracellular ActRIIa variant described herein.

[0105] I. Extracellular activin receptor type IIa (ActRIIa) variants Activin type II receptor is a single transmembrane domain receptor that regulates the signal of ligands in the transforming growth factor β (TGF-β) superfamily. Ligands in the TGF-β superfamily are involved in many physiological processes, such as muscle growth, blood vessel growth, cell differentiation, homeostasis and osteogenesis. Examples of ligands in the TGF-β superfamily include, for example, activins, inhibins, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin) and bone morphogenetic proteins (BMPs) (e.g., BMP9). Activins are abundantly expressed in bone tissue and regulate bone formation by controlling osteoblast and osteoclast function. Activins have been reported to be upregulated in bone diseases and inhibit osteoblast activity. Myostatin is also involved in bone homeostasis by increasing osteogenesis and inhibiting osteoblast activity. These data suggest that activin receptor ligands (e.g., activin and myostatin) promote bone resorption, which may lead to diseases or conditions involving bone damage, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss. Therefore, methods of reducing or inhibiting this signaling may be useful in treating diseases and conditions involving bone damage.

[0106] There are two types of activin type II receptors: ActRIIa and ActRIIb. Studies have shown that BMP9 binds to ActRIIb with a binding affinity about 300 times higher than ActRIIa (see, e.g., Townson et al., J. Biol. Chem. 287: 27313, 2012). ActRIIa is known to have a longer half-life than ActRIIb. The present invention describes an extracellular ActRIIa variant constructed by introducing the amino acid residues of ActRIIb into ActRIIa, the purpose of which is to confer the physiological properties conferred by ActRIIb while also maintaining the beneficial physiological and pharmacokinetic properties of ActRIIa. For example, the optimal peptide confer a significant increase in bone mineral density while maintaining a longer serum half-life and a low binding affinity to BMP9. Preferred ActRIIa variants also exhibit improved binding to activin and / or myostatin compared to wild-type ActRIIa, which allows them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling. These variants can be used to treat conditions in which activin receptor signaling is elevated, such as bone diseases, resulting in reduced bone resorption or osteoclast activity, and increased bone formation, bone mineral density, or bone strength. In some embodiments, amino acid substitutions can be introduced into the extracellular ActRIIa variants to reduce or eliminate the binding affinity of the variant to BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIa and ActRIIb are shown below.

[0107] Human ActRIIa, extracellular portion (SEQ ID NO: 73): Human ActRIIb, extracellular portion (SEQ ID NO: 74):

[0108] The polypeptides described herein include extracellular ActRIIa variants having at least one amino acid substitution relative to wild-type extracellular ActRIIa having a sequence of SEQ ID NO: 73 or an extracellular ActRIIa having a sequence of any one of SEQ ID NOs: 76-96. Possible amino acid substitutions at 27 different positions can be introduced into the extracellular ActRIIa variants (Table 1). In some embodiments, the extracellular ActRIIa variants can have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97% or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIa (SEQ ID NO: 73). The extracellular ActRIIa variant can have one or more (e.g., 1-27, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) amino acid substitutions relative to the sequence of wild-type extracellular ActRIIa (SEQ ID NO: 73). In some embodiments, the extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of SEQ ID NO: 1) can comprise amino acid substitutions at all 27 positions as listed in Table 1. In some embodiments, the extracellular ActRIIb variants can comprise amino acid substitutions at a number of positions, for example, at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 of the 27 positions as listed in Table 1.

[0109] Amino acid substitutions may deteriorate or improve the activity and / or binding affinity of the ActRIIa variants of the invention. To maintain polypeptide function, it is important to retain position X in the sequences shown in Tables 1 and 2 (SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). 17 Lysine (K) at position X. Substitution at this position can result in loss of activity. For example, an ActRIIa variant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCW LDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reduced in vivo activity, indicating that substitution of alanine (A) at position X is not tolerated. 17Thus, the ActRIIa variants of the invention, including the variants in Tables 1 and 2 (e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) have a lysine (K) at position X. 17 The amino acid K was retained.

[0110] The ActRIIa variants of the present invention preferably have reduced, weak or substantially no binding to BMP9. 23 , X 24 , X 25 and X 26 and maintaining the amino acid K at position X24 and having the amino acid K at position X24. 23 , X 24 , X 25 and X 26 In variants of the amino acid sequence TKEN at TEEN, BMP9 binding is reduced. The sequences TEEN and TKEN can be used interchangeably in ActRIIa variants of the invention (e.g., variants in Tables 1 and 2, e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to provide reduced BMP9 binding.

[0111] The ActRIIa variants of the present invention may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension may add one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) at the C-terminus to any of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). One potential C-terminal extension that may be included in the ActRIIa variants of the present invention is the amino acid sequence NP. For example, a sequence comprising a C-terminal extension is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). Another exemplary C-terminal extension that may be included in the ActRIIa variants of the present invention is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, a sequence comprising a C-terminal extension is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK).

[0112] Table 1. Amino acid substitutions in extracellular ActRIIa variants having the sequence of any one of SEQ ID NOs: 1-5

[0113] In some embodiments of the extracellular ActRIIa variant having the sequence of SEQ ID NO: 2, X3 Yes E, X 6 Yes R, X 11 It is D, X 12 It is K, X 13 Yes R, X 16 Is K or R, X 17 It is K, X 19 It is W, X 20 Yes L, X 21 is D, and X 22 is I or F. In some embodiments of the extracellular ActRIIa variant having the sequence of SEQ ID NO: 1 or 2, X 17 is K. In some embodiments of the extracellular ActRIIa variants having the sequence of SEQ ID NOs: 1-3, X 17 It is K, X 23 It's T, X 24 Yes E, X 25 is E, and X 26 is N. In some embodiments of the extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-5, X 17 It is K, X 23 It's T, X 24 It is K, X 25 is E, and X 26 It is N.

[0114] In some embodiments, a polypeptide described herein comprises an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 6-72 (Table 2).

[0115] Table 2. Extracellular ActRIIa variants having the sequences of SEQ ID NOs: 6-72

[0116] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIa variant (eg, any one of SEQ ID NOs: 1-72 (eg, SEQ ID NOs: 6-72)) has position X 17 The amino acid K at position X is changed 17For example, an ActRIIa variant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCW LDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reduced in vivo activity, indicating that substitution of position X by A is not tolerated. 17 K at.

[0117] In some embodiments, comprising a 23 , X 24 , X 25 and X 26 The polypeptide of the invention having an extracellular ActRIIa variant of the sequence TEEN at position (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be substituted with amino acid K at position X 24 In some embodiments, the amino acid E at position X 23 , X 24 , X 25 and X 26 The polypeptide of the invention having an extracellular ActRIIa variant of the sequence TKEN at position (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be substituted with amino acid E at position X 24 The amino acid K at position X 23 , X 24 , X 25 and X 26 Peptides with the sequences TEEN or TKEN at the position of the nucleotide sequence have reduced or weak binding to BMP9.

[0118] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIa variant (e.g., any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). In some embodiments, the C-terminal extension is the amino acid sequence NP. For example, the sequence comprising the C-terminal extension is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, the sequence comprising the C-terminal extension is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK). The C-terminal extension may add one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus.

[0119] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIa variant may further comprise a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) that may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant by way of a linker. In some embodiments, the moiety increases the stability of the polypeptide or improves the pharmacokinetic properties of the polypeptide. A polypeptide comprising an extracellular ActRIIa variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers that combine to form an Fc domain in a dimer.

[0120] In some embodiments, the extracellular ActRIIb variants described herein do not have the sequence of any of SEQ ID NOs: 76-96 shown in Table 3 below.

[0121] Table 3. Excluded extracellular ActRIIa variants.

[0122] In addition, in some embodiments, the polypeptides described herein have a serum half-life of at least 7 days in humans. The polypeptides can be expressed as D Binds to bone morphogenetic protein 9 (BMP9). The polypeptide can bind to bone morphogenetic protein 9 (BMP9) with a K of 10 pM or higher. DBinds to activin A. In some embodiments, the polypeptide does not bind to BMP9 or activin A. In some embodiments, the polypeptide binds to activin and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9. In some embodiments, the polypeptide having reduced or weak binding to BMP9 is at position X. 23 , X 24 , X 25 and X 26 The sequence TEEN or TKEN is present at the position.

[0123] Additionally, in some embodiments, the polypeptide can be expressed at a K of about 200 pM or more. D (e.g., a K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or more D , for example, a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 nM or more D , for example, a K of about 200 pM to about 50 nM D ) binds to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9. In some embodiments, the polypeptide can bind to human BMP9 with a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin A. In some embodiments, the polypeptide can bind to human activin A with a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin B. The polypeptide may also bind to human activin B with a K of about 5 pM or higher D (e.g., a K of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or more) D) binds to growth and differentiation factor 11 (GDF-11).

[0124] II. Fc domain In some embodiments, the polypeptides described herein may include an extracellular ActRIIa variant fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to increase the serum half-life of the polypeptide. A polypeptide comprising an extracellular ActRIIa variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers, which form an Fc domain in the dimer. As is conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain comprises two Fc domain monomers, which are bound by a C H 3 antibody constant domains and dimerize. The wild-type Fc domain forms the smallest structure that binds to Fc receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector functions, which is a typical "death" Fc domain. For example, the Fc domain can include specific amino acid substitutions known to minimize the interaction between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain is from an IgG1 antibody and includes amino acid substitutions L234A, L235A and G237A. In some embodiments, the Fc domain is from an IgG1 antibody and includes amino acid substitutions D265A, K322A and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The Kabat numbering of amino acid residues of a given antibody can be determined by aligning the homology regions of the antibody sequence with the "standard" Kabat numbering sequence. In addition, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a dimer of a polypeptide comprising an extracellular ActRIIa variant fused to an Fc domain monomer can be modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. The sequence of the Fc domain monomer that can be fused to the extracellular ActRIIa variant is shown below (SEQ ID NO: 97):

[0125] In some embodiments, the Fc domain is from an IgG1 antibody and comprises amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO: 97. In some embodiments, the Fc domain is from an IgG1 antibody and comprises amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO: 97. In some embodiments, the extracellular ActRIIa variants described herein (e.g., extracellular ActRIIa variants having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72))) can be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the Fc domain monomer. The Fc domain monomer can be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant.

[0126] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to an Fc domain. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. In some embodiments, the Fc domain contains a hinge domain. The Fc domain may be an Fc domain of an immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0127] Methods for engineering Fc domains with reduced dimerization are known in the art. In some embodiments, one or more amino acids with large side chains (e.g., tyrosine or tryptophan) can be introduced into the C H 3-C H 3 dimer interface to hinder dimer formation due to steric collision. In other embodiments, one or more amino acids with small side chains (e.g., alanine, valine, or threonine) can be introduced into the C H 3-C H 3 dimer interface to remove favorable interactions. HMethods for introducing amino acids with large or small side chains into the 3 domain are described, for example, in Ying et al. (J Biol Chem. 287: 19399-19408, 2012), U.S. Patent Publication No. 2006 / 0074225, U.S. Patent Nos. 8,216,805 and 5,731,168, Ridgway et al. (Protein Eng. 9: 617-612, 1996), Atwell et al. (J Mol Biol. 270: 26-35, 1997), and Merchant et al. (Nat Biotechnol. 16: 677-681, 1998), all of which are incorporated herein by reference in their entirety.

[0128] In yet other embodiments, the C segment between the two Fc domains H 3-C H 3. Interface C H One or more amino acid residues in the C3 domain are replaced with a positively charged amino acid residue (e.g., lysine, arginine, or histidine) or a negatively charged amino acid residue (e.g., aspartic acid or glutamic acid) such that the interaction becomes electrostatically unfavorable, depending on the specific charged amino acid introduced. H Methods for introducing charged amino acids into the 3 domain to disfavor or prevent dimer formation are described, for example, in Ying et al. (J Biol Chem. 287: 19399-19408, 2012), U.S. Patent Publication Nos. 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0129] In some embodiments of the invention, the Fc domain comprises one or more of the following amino acid substitutions relative to the sequence of human IgG1: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T and K409I. In a specific embodiment, the Fc domain comprises the amino acid substitution T366W relative to the sequence of human IgG1. The sequence of the wild-type Fc domain is shown in SEQ ID NO: 151.

[0130] III. Albumin Binding Peptides In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to a serum protein binding peptide. Binding to a serum protein peptide may improve the pharmacokinetics of a protein drug.

[0131] As an example, albumin binding peptides useful in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0132] In the present invention, the albumin binding peptide can be linked to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) to increase the serum half-life of the extracellular ActRIIa variant. In some embodiments, the albumin binding peptide is linked directly or through a linker to the N- or C-terminus of the extracellular ActRIIa variant.

[0133] In some embodiments, the extracellular ActRIIa variants described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of an albumin binding peptide (e.g., SEQ ID NO: 152) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the albumin binding peptide. Without being bound by theory, it is expected that the inclusion of an albumin binding peptide in the extracellular ActRIIa variants described herein can result in prolonged retention of the therapeutic protein through its binding to serum albumin.

[0134] IV. Fibronectin Domain In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to a fibronectin domain. Binding to the fibronectin domain may improve the pharmacokinetics of protein drugs.

[0135] The fibronectin domain is a high molecular weight glycoprotein or fragment thereof of the extracellular matrix that binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, the fibronectin domain is connected to the N- or C-terminus (e.g., C-terminus) of an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) to increase the serum half-life of the extracellular ActRIIa variant. The fibronectin domain can be connected directly or via a linker to the N- or C-terminus of an extracellular ActRIIa variant.

[0136] As an example, fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: 153) having amino acids 610-702 of the sequence of UniProt ID NO: P02751. In another embodiment, the fibronectin domain is an adnectin protein.

[0137] In some embodiments, the extracellular ActRIIa variants described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of a fibronectin domain (e.g., SEQ ID NO: 153) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the fibronectin domain. Without being bound by theory, it is expected that the inclusion of a fibronectin domain in the extracellular ActRIIa variants described herein can result in prolonged retention of the therapeutic protein through its binding to integrins and extracellular matrix components (such as collagen and fibrin).

[0138] V. Serum albumin In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to serum albumin. Binding to serum albumin may improve the pharmacokinetics of protein drugs.

[0139] Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and accounts for about half of serum proteins. It is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids and other proteins in the body, buffering pH, and maintaining the osmotic pressure required for the correct distribution of body fluids between blood vessels and body tissues. In a preferred embodiment, serum albumin is human serum albumin. In some embodiments of the present invention, human serum albumin is connected to the extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) N- or C-terminus (e.g., C-terminus) to increase the serum half-life of the extracellular ActRIIa variant. Human serum albumin can be directly or via a linker connected to the N- or C-terminus of the extracellular ActRIIa variant.

[0140] As an example, serum albumin useful in the methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0141] In some embodiments, the extracellular ActRIIa variants described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of human serum albumin (e.g., SEQ ID NO: 154) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the serum albumin. Without being bound by theory, it is expected that the inclusion of serum albumin in the extracellular ActRIIa variants described herein can result in prolonged retention of the therapeutic protein.

[0142] VI. Connectors The polypeptides described herein may comprise an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety by means of a linker. In some embodiments, the moiety increases the stability of the polypeptide. Exemplary moieties include an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin. In the present invention, the linker between a moiety (e.g., an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., SEQ ID NO: 151), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide (e.g., SEQ ID NO: 152), a fibronectin domain (e.g., SEQ ID NO: 153, or human serum albumin (e.g., SEQ ID NO: 154)) and an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be an amino acid spacer comprising 1-200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the spacer region may contain a motif, such as a multiple or repeated motif, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO:98), GGGS (SEQ ID NO:99), GGGG (SEQ ID NO:100), GGGGA (SEQ ID NO:101), GGGGS (SEQ ID NO:102), GGGGG (SEQ ID NO:103), GGAG (SEQ ID NO:104), GGSG (SEQ ID NO:105), AGGG (SEQ ID NO:106), or SGGG (SEQ ID NO:107).In some embodiments, the spacer region can contain 2 to 12 amino acids, including GA or GS, such as the motifs of GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer region can contain 3 to 12 amino acids, including GGA or GGS, such as the motifs of GGA, GGS, GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). In still other embodiments, the spacer can contain 4 to 12 amino acids, including a motif of GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), e.g., GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In some embodiments, the spacer can contain a motif of GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128), and GGGGSGGGGSGGGGS (SEQ ID NO: 129).In some embodiments of the invention, the linker between the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or serum albumin) and the extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

[0143] In some embodiments, the spacer can also contain amino acids other than glycine, alanine and serine, such as AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), In some embodiments, the spacer may contain a motif, such as multiple or repeated motifs, of EAAAK (SEQ ID NO: 147). In some embodiments, the spacer may contain a proline-rich sequence, such as (XP) n ), such as a multiple or repeating motif, wherein X can be any amino acid (e.g., A, K or E) and n is 1-5, and PAPAP (SEQ ID NO: 148).

[0144] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0145] VII. Vectors, Host Cells and Protein Production The polypeptide of the present invention can be produced from a host cell. A host cell refers to a medium comprising essential cellular components (e.g., organelles) required for expressing a polypeptide described herein and a fusion polypeptide from its corresponding nucleic acid. Nucleic acid can be included in a nucleic acid vector, which can be introduced into a host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, injection, etc.). The selection of nucleic acid vectors depends in part on the host cell to be used. Typically, a preferred host cell is a eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) source.

[0146] Nucleic acid vector construction and host cells The nucleic acid sequence encoding the amino acid sequence of the polypeptide of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptide of the present invention can be obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques in the art (e.g., QuikChange TM Mutagenesis) mutates a nucleic acid molecule encoding wild-type extracellular ActRIIa to include a specific amino acid substitution. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0147] The nucleotide sequence encoding the polypeptide of the present invention can be inserted into a vector that can replicate and express nucleic acid molecules in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for purposes of the present invention. Every kind of vector can include various components that can be adjusted and optimized for compatibility with a specific host cell. For example, vector components can include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleotide sequence encoding a target protein, and a transcription termination sequence.

[0148] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells. In some embodiments, E. coli cells can also be used as host cells of the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 ( 31,446), Escherichia coli λ1776( 31,537, Escherichia coli BL21(DE3) ( BAA-1025), and Escherichia coli RV308 ( 31,608). Different host cells have characteristics and specific mechanisms for post-transcriptional processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed polypeptide. The above expression vectors can be introduced into appropriate host cells using conventional techniques in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). Once the vector is introduced into the host cell for protein production, the host cell is cultured in a conventional nutrient medium appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequence. Methods for expressing therapeutic proteins are known in the art, see, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd edition 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd edition 2012.

[0149] Protein production, recovery and purification The host cells used to produce the polypeptides of the present invention can be grown in a medium known in the art and suitable for culturing the selected host cells. Examples of suitable culture media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293 TM Expression medium, DMEM supplemented with fetal bovine serum (FBS) and RPMI-1640. Examples of suitable culture media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as selection agents, for example ampicillin. The host cells are grown at a suitable temperature such as about 20°C to about 39°C, for example 25°C to about 37°C, preferably 37°C, and CO 2 The pH of the culture medium is generally about 6.8 to 7.4, for example 7.0, which depends mainly on the host organism. If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter.

[0150] In some embodiments, depending on the expression vector and host cell used, the expressed protein can be secreted from the host cell (e.g., mammalian host cell) into the cell culture medium. Protein recovery can involve filtering the cell culture medium to remove cell debris. The protein can be further purified. The polypeptide of the present invention can be purified by any method known in the field of protein purification, for example, by chromatography (e.g., ion exchange, affinity and size exclusion column chromatography), centrifugation, differential solubility or by any other standard technique for protein purification. For example, the protein can be separated and purified by appropriately selecting and combining affinity columns, such as protein A columns (e.g., POROS protein A chromatography) and chromatographic columns (e.g., POROS HS-50 cation exchange chromatography), filtering, ultrafiltration, salting out and dialysis procedures.

[0151] In other embodiments, the host cells can be destroyed, for example, by osmotic shock, ultrasonic treatment or lysis to destroy the host cells to recover the expressed protein. Once the cells are destroyed, the cell debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be conjugated to a marker sequence (such as a peptide) to facilitate purification. An example of a marker amino acid sequence is a six-histidine peptide (His-tag), which binds to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin "HA" tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., Cell 37:767, 1984).

[0152] Alternatively, the polypeptides of the invention can be produced by cells of a subject (e.g., a human), for example, in the context of gene therapy, by administering a vector (such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxviral vector (e.g., a vaccinia virus vector, such as modified vaccinia virus Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding the polypeptides of the invention. The vector, once within the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), will promote expression of the polypeptide, which is then secreted from the cells. If treatment of the disease or condition is the desired outcome, no further action may be required. If it is desired to collect the protein, blood can be collected from the subject and the protein purified from the blood by methods known in the art.

[0153] VIII. Pharmaceutical Compositions and Formulations The invention features a pharmaceutical composition comprising a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, a pharmaceutical composition of the invention comprises a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the invention comprise a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a portion (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or serum albumin) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the invention comprising the polypeptides of the invention may be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions for therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients, which may be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical compositions of the invention comprise a nucleic acid molecule (DNA or RNA, such as mRNA) encoding a polypeptide of the invention, or a vector containing such a nucleic acid molecule.

[0154] Acceptable carrier and excipient in the pharmaceutical composition are nontoxic to receptors at dosage and concentration used.Acceptable carrier and excipient can include buffer, such as phosphate, citrate, HEPES and TAE, antioxidant, such as ascorbic acid and methionine, preservative, such as hexamethylammonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol and benzalkonium chloride, protein, such as human serum albumin, gelatin, dextran and immunoglobulin, hydrophilic polymer, such as polyvinyl pyrrolidone, amino acid, such as glycine, glutamine, histidine and lysine, and carbohydrate, such as glucose, mannose, sucrose and sorbitol.The pharmaceutical composition of the present invention can be administered parenterally in the form of injectable preparations.A sterile solution or any pharmaceutically acceptable liquid can be used to prepare a pharmaceutical composition for injection as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture medium (e.g., Dulbecco's modified Eagle medium (DMEM), α-modified Eagles medium (α-MEM), F-12 medium). Formulation methods are known in the art, see, e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd edition) Taylor & Francis Group, CRC Press (2015).

[0155] The pharmaceutical composition of the present invention can be prepared in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules. The pharmaceutical composition of the present invention can also be prepared in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules. Such technology is described in Remington: The Science and Practice of Pharmacy 22nd Edition (2012). The pharmaceutical composition to be used for in vivo administration must be sterile. This can be easily accomplished by filtering through a sterile filtration membrane.

[0156] The pharmaceutical composition of the present invention can also be prepared as a sustained release formulation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide of the present invention. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM Some sustained release formulations enable release of the molecule over several months, for example one to six months, while other formulations release the pharmaceutical composition of the invention for shorter periods of time, for example days to weeks.

[0157] The pharmaceutical composition can be formed in unit dosage form as desired. The amount of active ingredient (eg, polypeptide of the present invention) included in the pharmaceutical preparation is such as to provide a suitable dosage within the specified range (eg, a dosage within the range of 0.01-100 mg / kg body weight).

[0158] The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can include a sustained-release matrix wherein a gene delivery vector is embedded. If hydrodynamic injection is used as a delivery method, the pharmaceutical composition containing nucleic acid molecules encoding polypeptides as described herein or the carrier (e.g., viral vector) containing the nucleic acid molecules containing the nucleic acid molecules is delivered intravenously and rapidly with a large fluid volume. The carrier that can be used as a gene delivery vehicle in vivo includes but is not limited to retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified vaccinia virus Ankara), adeno-associated virus vectors and alphavirus vectors.

[0159] IX. Routes, Dosages, and Administration Pharmaceutical compositions comprising polypeptides of the present invention as therapeutic proteins can be formulated for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical compositions can also be formulated for oral, nasal, spray, aerosol, rectal, or vaginal administration or administered via oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective drug carriers are known in the art. See, for example, ASHP Handbook on Injectable Drugs, Toissel, 18th edition (2014).

[0160] In some embodiments, a pharmaceutical composition comprising a nucleic acid molecule encoding a polypeptide of the present invention or a vector containing such a nucleic acid molecule can be administered by gene delivery. The method of gene delivery is well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified vaccinia virus Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the mRNA molecule encoding the polypeptide of the present invention can be directly administered to a subject.

[0161] In some embodiments of the present invention, the nucleic acid molecules encoding polypeptides described herein or the carriers containing such nucleic acid molecules can be administered using a fluid dynamic injection platform. In the fluid dynamic injection method, the nucleic acid molecules encoding polypeptides described herein are placed under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). Plasmids are often delivered intravenously and rapidly with a large fluid volume. The fluid dynamic injection uses the controlled fluid dynamic pressure in the vein to enhance cell permeability, so that the pressure from the rise of the rapid injection of a large fluid volume causes fluid and plasmid to extravasate from the vein. The expression of nucleic acid molecules is mainly driven by the liver. In mice, fluid dynamic injection is often performed by injecting plasmids into the tail vein. In certain embodiments, the mRNA molecules encoding polypeptides described herein can be administered using fluid dynamic injection.

[0162] The dosage of the pharmaceutical composition of the present invention depends on factors, including route of administration, disease to be treated and physical characteristics of the subject, such as age, weight, general health. The pharmaceutical composition of the present invention can include a dosage of the polypeptide of the present invention ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg / kg), and in a more specific embodiment, about 0.1 to about 30 mg / kg, and in a more specific embodiment, about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors, such as the degree of the disease and different parameters of the subject.

[0163] The pharmaceutical composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount that results in the improvement or remedy of symptoms. The pharmaceutical composition is administered in various dosage forms, such as intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Typically, therapeutic proteins are administered at 0.1-100 mg / kg, such as 1-50 mg / kg. The pharmaceutical composition comprising the polypeptide of the present invention can be administered to a subject in need thereof, for example, every day, every week, every two weeks, every month, every two months, every quarter, every two years, once or multiple times per year (e.g., 1-10 times or more), or when medically necessary. In some embodiments, the pharmaceutical composition comprising the polypeptide of the present invention can be administered to a subject in need thereof weekly, every two weeks, every month, every two months, or every quarter. The dosage can be provided in a single dose or multiple dose regimen. As the patient's health condition declines, as medical conditions improve or increase, the timing between administrations can be reduced.

[0164] X. Treatment Methods The present invention is based on the discovery that substitution of amino acids from the extracellular portion of ActRIIb into the extracellular portion of ActRIIa produces ActRIIa variants with improved properties. ActRIIa variants generated by introducing residues from ActRIIb into ActRIIa retain the beneficial properties of ActRIIa, such as longer serum half-life and low binding affinity to BMP9, and acquire some of the beneficial properties of ActRIIb, such as increased binding to activin A and B (see Table 4). These ActRIIa variant properties produce polypeptides that can be used therapeutically to compete with endogenous activin receptors for ligand binding. Because ActRIIa variants contain the extracellular portion of the receptor, they are soluble and are able to bind and sequester ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Thus, extracellular ActRIIa variants can be used to treat diseases or conditions in which elevated activin signaling has been implicated (e.g., associated with increased expression of an activin receptor or an activin receptor ligand). For example, activin has been found to be upregulated in bone disease and is known to inhibit osteoblast activity, suggesting that increased activin levels lead to bone disease. Thus, treatment with a therapeutic agent that binds activin and reduces its interaction with endogenous receptors can be used to increase bone mineral density and treat subjects with diseases or conditions involving bone damage.

[0165] The present invention provides compositions and methods of treating that can be used to increase bone mineral density, increase bone formation, or reduce bone resorption in a subject in need thereof. In some embodiments, the subject may have a disease that causes bone damage (e.g., osteoporosis or osteopenia). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling in a subject with a disease or condition involving bone damage. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant as described herein reduces or inhibits the binding of myostatin, activin, and / or BMP9 to its receptor, such as ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). In some embodiments, affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to its receptor, such as ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa)) results in an increase in bone mineral density or bone formation in the subject or a decrease in bone resorption in the subject.

[0166] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be administered to a subject to increase bone mineral density, increase bone formation, or decrease bone resorption, or affect myostatin, activin, and / or BMP9 signaling in the subject. In some embodiments, the methods described herein increase bone mineral density in the subject. In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage. In some embodiments of the methods described herein, the subject has a disease or condition involving bone damage (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss).

[0167] The present invention also includes methods of treating a subject having primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss by administering to the subject a polypeptide described herein, e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, the primary osteoporosis is age-related or hormone-related osteoporosis (e.g., associated with estrogen decline). In some embodiments, the secondary osteoporosis is immobilization-induced or glucocorticoid-induced osteoporosis. In some embodiments, the bone cancer is multiple myeloma or the cancer metastasis-related bone loss is caused by multiple myeloma. In some embodiments, the treatment-related bone loss occurs due to treatment with FGF-21 or GLP-1, treatment with a therapeutic agent containing FGF-21 or GLP-1, or treatment of type 2 diabetes and / or obesity, or due to cancer therapy (e.g., chemotherapy or radiation therapy). In some embodiments, the diet-related bone loss is rickets (e.g., vitamin D deficiency). In some embodiments, the hypogravity-related bone loss lacks load-related bone loss.

[0168] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be used to prevent the development of a disease or condition involving bone damage (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, osteosclerosis, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss) and / or treat a patient who has been diagnosed with a disease or condition involving bone damage. A polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be prophylactically administered to a patient who is likely to develop a disease or condition involving bone damage, e.g., an individual with a genetic predisposition, family history, of bone damage or low bone mass, such that the extracellular ActRIIa polypeptide can prevent or delay the development of bone damage.

[0169] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be administered to a subject to prevent the development of a disease or condition involving bone damage (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, obesity therapy-related bone loss, low gravity-related bone loss, or immobility-related bone loss) and / or treat a disease or condition involving bone damage. or conditions (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss), or affecting myostatin, activin, and / or BMP9 signaling in a subject (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to its receptor). In some embodiments, the methods described herein increase bone mineral density (e.g., increase bone mass). In some embodiments, the methods described herein reduce bone resorption (e.g., reduce bone catabolic activity). In some embodiments, the methods described herein increase bone formation (e.g., increase bone anabolic activity or increase bone generation). In some embodiments, the methods described herein increase osteoblast activity or osteoblastogenesis. In some embodiments, the methods described herein reduce osteoclast activity or osteoclastogenesis. In some embodiments, the methods described herein reduce or inhibit the binding of activin and / or myostatin to its receptor. In some embodiments, the methods increase bone formation, increase bone mineral density, or decrease bone resorption in cortical or trabecular bone.

[0170] In any of the methods described herein, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs: 6-71)) further comprising a C-terminal extension of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6 or more amino acids) can be used as a therapeutic protein. In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs: 6-71)) fused to an Fc domain monomer can be used as a therapeutic protein. In any of the methods described herein, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a portion (e.g., a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) can be used as a therapeutic protein. Nucleic acids encoding polypeptides described herein or vectors containing the nucleic acids can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptides, nucleic acids, or vectors can be administered as part of a pharmaceutical composition. Example

[0171] Example 1 - Assessment of ActRIIa variant binding affinity by surface plasmon resonance (SPR) Biacore 3000 was used to measure the kinetics of the interaction between ActRIIa variants and the ligands activin A, activin B, growth differentiation factor 11 (GDF11), and BMP-9. ActRIIa variants were transiently expressed in HEK293 cells and purified from conditioned media using protein-A sepharose chromatography. ActRIIa variants were immobilized on the chip (CM4 or CM5) in flow chambers 2-4 with capture antibodies (anti-mouse from GEGE) to ensure correct orientation. Flow chamber 1 was used as a reference chamber to subtract any nonspecific binding and volume effects. From GE Healthcare TM HBS-EP+ buffer was used as running buffer. Each ligand was run in a concentration series at 40 μl / min to avoid mass transfer effects. TM The Scrubber2 software analyzed the data to calculate the K for each interaction. D (Table 4).

[0172] Table 4: Binding affinity (K) of ActRIIa variants to various ligands D )

[0173] Example 2 - Effects of Extracellular ActRIIa Variants on Bone Mineral Density Adult male C57 / BL6 mice received sham surgery (SHAM) or castration-surgery (ORX). Both surgical groups were allowed to recover for 14 days after surgery. All animals were housed in conventional cages with free access to food (regular food) and water. SHAM and ORX animals were then assigned to vehicle-treated groups (VEH) or ActRII variant-treated groups and received biweekly systemic intraperitoneal administration of vehicle or ActRII variant (10 mg / kg) for 71 days. Body weight was measured twice a week during treatment. Body composition was analyzed using a MiniSpec LF50 NMR analyzer on day 0 of the study and then on days 14, 28, 47, and 71 after the start of treatment. At the study termination date, target tissues (muscle, fat depots, and tibia) were surgically removed, weighed, and appropriately stored for further analysis. At this time, ORX animals were also examined to confirm complete removal of the testicles. After the termination of the experiment, micro-computed tomography was also used to evaluate the cortical morphometrics and trabecular structure of various bones.

[0174] Other Implementations Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention which follow from the principles of the invention in general and include such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and which may be applied to the basic features herein above described.

[0175] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0176] Other implementations are within the following claims.

Claims

1. A polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant having the following sequence: GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX17X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKF SYFPEMEVTQPTS(SEQ IDNO:1), Where X1 is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 It is K or Q; X 11 It is D or A; X 12 It is K or A; X 13 is R or A; X 14 is R or L; X 15 is F or Y; X 16 is K, R or A; X 17 is K, A, Y, F or I; X 18 Q or K; X 19 W or A; X 20 L or A; X 21 is D, K, R, A, F, G, M, N, or I; X 22 is I, F or A; X 23 is K or T; X 24 Is K or E; X 25 Is D or E; X 26 is S or N; and X 27 is E or Q, and wherein the variant has at least one amino acid substitution relative to wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or extracellular ActRIIa having any one of the sequences of SEQ ID NOs: 76-96.

2. The polypeptide of claim 1, wherein the variant has the following sequence: GAILGRSETQECLFX2NANWX3X4X5X6TNQTGVEX7CX8GX9KX 11 X 12 X 13 X 14 HCX 15 ATWX 16 NOT SGSIEIVX17X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSY FPEMEVTQPTS(SEQ IDNO:2)。 3. The polypeptide of claim 1 or 2, wherein the variant has the following sequence: GAILGRSETQECLFX2NANWEX4X5RTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIE IVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQ PTS(SEQ ID NO:3)。 4. The polypeptide of any one of claims 1 to 3, wherein the variant has the following sequence: GAILGRSETQECLFX2NANWEX4DRTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEI VKX 18 GCWLDDX 22 NCYDRTDCVEX 23 Kx 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQP TS(SEQ ID NO:4)。 5. The polypeptide of any one of claims 1 to 4, wherein the variant has the following sequence: GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14 HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO:5)。 6. The polypeptide of claim 1, wherein X1 is F.

7. The polypeptide of claim 1, wherein X1 is Y.

8. The polypeptide of claim 1, 6 or 7, wherein X 10 It's K.

9. The polypeptide of claim 1, 6 or 7, wherein X 10 It's Q.

10. The polypeptide of any one of claims 1 to 9, wherein X2 is F.

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