Pharmaceutical composition containing activin type IIA receptor variant

Abstract

To provide an activin type IIA receptor variant and a method for using thereof.SOLUTION: The present invention is characterized by a polypeptide containing an extracellular ActRIIA variant. In some embodiments, the polypeptide of the present invention contains an extracellular ActRIIA variant fused to a monomer or a portion of Fc domain. The present invention is also characterized by a pharmaceutical composition and method using the polypeptide in order to treat a disease and a disease state including low red blood cell level (e.g., anemia or blood loss), fibrosis, or pulmonary hypertension.SELECTED DRAWING: Figure 2

Description

[Technical Field]

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

[0002] Fibrosis is the formation of excess connective tissue in organs and tissues. Connective tissue, which can form in response to damage (e.g., injury) or as part of an immune response (e.g., inflammatory response), can disrupt the structure and function of the organ or tissue in which it is formed and increase tissue stiffness. Fibrosis can occur in many organs and tissues in the body, including the lungs (e.g., pulmonary fibrosis, cystic fibrosis), liver (e.g., cirrhosis), heart (e.g., endomyocardial fibrosis or post-myocardial infarction fibrosis), brain (e.g., glial scar formation), skin (e.g., keloid formation), kidneys (e.g., renal fibrosis), and eyes (e.g., corneal fibrosis), among others, and is known to be associated with certain medical treatments (e.g., chemotherapy, radiation therapy, and surgery). Treatment options for patients with fibrosis are limited, with most treatments focused on improving quality of life or temporarily slowing disease progression.

[0003] Anemia is a global health problem with health consequences that affect both morbidity and mortality. In the United States alone, the prevalence of anemia nearly doubled from 2003 to 2012. Symptoms of anemia include fatigue, weakness, shortness of breath, palpitations, and decreased cognitive ability. Children, pregnant women, women of reproductive age, and the elderly are at highest risk of developing anemia. The most common form of anemia is iron deficiency anemia, but anemia can also be caused by chronic disease, blood loss, and red blood cell destruction. While iron deficiency anemia can be treated with iron supplements, many other forms of anemia, such as aplastic anemia, anemia of chronic disease, and hemolytic anemia, may require blood transfusions.

[0004] Pulmonary hypertension (PH) is a serious condition characterized by higher-than-normal pressure in the blood vessels between the lungs and heart. PH can be classified into five major types, also known as WHO Groups I–V: arterial (PAH), venous (PH secondary to left-sided heart disease), hypoxic (PH caused by lung disease), thromboembolic (PH caused by chronic arterial obstruction, such as a blood clot), or other (PH with unknown mechanisms or multifactorial causes). PAH is characterized by elevated pulmonary vascular pressure due to blockage or narrowing of small blood vessels in the lungs caused by scarring. This increases resistance to blood flow through the lungs and forces the right side of the heart to work harder, potentially leading to heart failure, reduced blood oxygenation, and shortened life expectancy. PAH can be idiopathic (e.g., without an identifiable cause), hereditary (e.g., familial, often due to a genetic mutation), or associated with drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital heart abnormalities, and connective tissue / autoimmune diseases (such as scleroderma and lupus). Treatments for PH include vasodilators, anticoagulants, and supplemental oxygen; however, these treatments manage the symptoms of the disease rather than target the underlying biological mechanisms. Summary of the Invention [Problem to be solved by the invention]

[0005] New treatments for fibrosis, anemia, and PH are needed. [Means for solving the problem]

[0006] The present invention relates to polypeptides comprising an extracellular activin receptor type IIA (ActRIIA) variant. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIA variant fused to the N- or C-terminus of an Fc domain monomer or moiety. Such moieties may be attached by amino acids or other covalent bonds and increase the stability of the polypeptide. Polypeptides comprising an extracellular ActRIIA variant fused to an Fc domain monomer may also form dimers (e.g., homodimers or heterodimers) through interaction between two Fc domain monomers. The polypeptides of the present invention may be used to reduce or prevent fibrosis, or to slow or inhibit the progression of fibrosis in subjects with or at risk of developing fibrosis. Polypeptides of the invention can also be used, for example, to increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, and / or increase red blood cell count, e.g., increase red blood cell mass) or to increase red blood cell formation in a subject in need thereof (e.g., a subject having or at risk of developing low red blood cell levels (e.g., low hemoglobin levels, low hematocrit, and / or low red blood cell count (e.g., low red blood cell mass)), e.g., anemia or blood loss). Furthermore, polypeptides of the invention can be used to treat, prevent, delay, or reduce the onset or progression of pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or other pulmonary hypertension) in a subject having or at risk of developing pulmonary hypertension. Additionally, the polypeptides of the present invention can also be used to affect myostatin, activin, and / or bone morphogenetic protein 9 (BMP9) signaling in subjects with or at risk of developing fibrosis, low red blood cell levels (e.g., low hemoglobin levels, low hematocrit, and / or low red blood cell count (e.g., low red blood cell mass)), or pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or other pulmonary hypertension).

[0007] In one aspect, the present invention provides a polypeptide comprising an extracellular activin type IIA receptor (ActRIIA) variant, said variant comprising: GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 is K or Q;X 11 is D or A;X 12 is K or A;X 13 is R or A; X 14 is R or L; X 15 is F or Y;X 16 is K, R, or A; X 17 is K, A, Y, F, or I; X 18 is Q or K;X 19 is W or A;X 20 is L or A;X 21 is D, K, R, A, F, G, M, N, or I; X 22 is I, F, or A; X 23 is K or T;X 24 is K or E;X 25 is D or E;X 26is S or N; and X 27 is E or Q, and the mutant relates to a polypeptide having at least one amino acid substitution relative to a wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73 or an extracellular ActRIIA having any one of the sequences of SEQ ID NOs: 76 to 96.

[0008] In some embodiments, the variant is GAILGRSETQECLFX2NANWX3X4X5X6TNQTGVEX7CX8GX9KX 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 2), wherein X2, X3, X4, X5, X6, X7, X8, X9, X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , and X 27 is defined in the same way as above.

[0009] In some embodiments, the variant is: GAILGRSETQECLFX2NANWEX4X5RTNQTGVEX7CX8GX9KDKRX 14 HCX15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3), wherein X2, X4, X5, X7, X8, X9, X 14 , X 15 , X 16 , X 18 , X 22 , X 23 , X 24 , X 25 , X 26 , and X 27 is defined as above.

[0010] In some embodiments, the variant is: GAILGRSETQECLFX2NANWEX4DRTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4), wherein X2, X4, X7, X8, X9, X 14 , X 15 , X 16 , X 18 , X 22 , X 23 , X 25 , X 26 , and X 27 is defined as above.

[0011] In some embodiments, the variant is: GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5), wherein X2, X4, X8, X9, X 14 , X 18 , X 23 , X 25 , X 26 , and X 27 is defined as above.

[0012] In any of the above embodiments, X1 is F or Y. In any of the above embodiments, X2 is F or Y. In any of the above embodiments, X3 is E or A. In any of the above embodiments, X4 is K or L. In any of the above embodiments, X5 is D or E. In any of the above embodiments, X6 is R or A. In any of the above embodiments, X7 is P or R. In any of the above embodiments, X8 is Y or E. In any of the above embodiments, X9 is D or E. In any of the above embodiments, X 10 is K or Q. In any of the above embodiments, X 11 is D or A. In any of the above embodiments, X 12 is K or A. In any of the above embodiments, X 13 is R or A. In any of the above embodiments, X 14 is R or L. In any of the above embodiments, X 15 is F or Y. In any of the above embodiments, X 16 is K, R, or A. In any of the above embodiments, X 17 is K, A, Y, F, or I. In any of the above embodiments, X 18 is Q or K. In any of the above embodiments, X 19is W or A. In any of the above embodiments, X 20 is L or A. In any of the above embodiments, X 21 is D, K, R, A, F, G, M, N, or I. In any of the above embodiments, X 22 is I, F, or A. In any of the above embodiments, X 23 is K or T. In any of the above embodiments, X 24 is K or E. In any of the above embodiments, X 25 is D or E. In any of the above embodiments, X 26 is S or N. In any of the above embodiments, X 27 is E or Q. In any of the above embodiments, X 23 is T and X 24 is E and X 25 is E and X 26 is N. In any of the above embodiments, X 23 is T and X 24 is K and X 25 is E and X 26 is N. In any of the above embodiments, X 17 is K.

[0013] In any of the above embodiments, the mutant has the sequence of any one of SEQ ID NOs: 6-72. In any of the above embodiments, position X 24 The amino acid can be substituted with the amino acid K.

[0014] In any of the above embodiments, position X 24 can be substituted with the amino acid E. In any of the above embodiments, the polypeptides described herein may further comprise a C-terminal extension of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids). In some embodiments, the C-terminal extension is the amino acid sequence NP. In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 155).

[0015] In any of the above-described embodiments, the polypeptides described herein may further comprise a moiety fused or covalently bound to the C-terminus of the polypeptide. In some embodiments, the moiety increases the stability or improves the pharmacokinetics of the polypeptide. In some embodiments, the moiety is an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin.

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

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

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

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

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

[0021] In some embodiments, the linker is an amino acid spacer, hi some embodiments, the amino acid spacer is GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

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

[0023] In any of the above embodiments, the polypeptides described herein have a serum half-life of at least 7 days (eg, in a human subject). In any of the above embodiments, the polypeptides described herein have a K D In some embodiments, the polypeptide binds to human bone morphogenetic protein 9 (BMP9) at a specific binding site. In some embodiments, the polypeptide binds to activin and / or myostatin, and exhibits low (e.g., weak) binding to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0024] In any of the above embodiments, the polypeptides described herein have a K D It binds to human activin A at In any of the above embodiments, the polypeptides described herein have a K D It binds to human activin B at

[0025] In any of the above embodiments, the polypeptides described herein have a K D It binds to human GDF-11. In another aspect, the present invention relates to a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA mutant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). In another aspect, the present invention also relates to a vector comprising the nucleic acid molecule described herein.

[0026] In another aspect, the invention relates to a host cell expressing a polypeptide as described herein, which host cell comprises a nucleic acid molecule or vector as described in the previous two aspects, which nucleic acid molecule or vector is expressed in the host cell.

[0027] In another aspect, the present invention relates to a method of making a polypeptide as described herein, the method comprising: a) providing a host cell comprising a nucleic acid molecule or vector as described herein; and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow the formation of the polypeptide.

[0028] In another aspect, the invention relates to a pharmaceutical composition comprising a polypeptide, nucleic acid molecule, or vector described herein and one or more pharmaceutically acceptable carriers or excipients, in some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or vector is in a therapeutically effective amount.

[0029] In another aspect, the present invention also relates to a construct (e.g., a homodimer) comprising two identical polypeptides, each comprising an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72), fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NOS: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0030] In another aspect, the present invention also relates to a construct comprising two different polypeptides (e.g., heterodimers) each comprising an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72), fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NOS: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0031] In another aspect, the present invention relates to a method of reducing or preventing fibrosis in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0032] In another aspect, the present invention relates to a method of slowing, inhibiting, or reversing the progression of fibrosis in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0033] In another aspect, the present invention relates to a method of reducing the risk of developing fibrosis or ameliorating existing fibrosis in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0034] In another aspect, the invention relates to a method of treating a subject having or at risk of developing fibrosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0035] In another aspect, the invention relates to a method of reducing the development of fibrosis by administering to a subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0036] In another aspect, the invention relates to a method of reversing fibrosis by administering to a subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0037] In another aspect, the present invention relates to a method for affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their receptors) in a subject having or at risk of developing fibrosis, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0038] In some embodiments of any of the above aspects, the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, bone marrow fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, osteoarthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid. In some embodiments, the tissue fibrosis is fibrosis affecting a tissue selected from the group consisting of muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine, large intestine, biliary tract, and intestine.

[0039] In some embodiments of any of the above aspects, the fibrosis is fibrosis associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments of any of the above aspects, the fibrosis is caused by chronic kidney disease.

[0040] In some embodiments of any of the above aspects, the method improves the function of a fibrotic tissue or organ. In some embodiments of any of the above aspects, the method slows, inhibits, or reverses the progression of fibrosis. In some embodiments of any of the above aspects, the method reduces (e.g., reduces the frequency or severity of) or reverses one or more symptoms of fibrosis.

[0041] In another aspect, the present invention relates to a method of increasing red blood cell levels (e.g., increasing hemoglobin levels, red blood cell count, or hematocrit, e.g., increasing red blood cell mass) in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0042] In another aspect, the present invention relates to a method of promoting or increasing red blood cell formation in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0043] In some embodiments of any of the above aspects, the subject suffers from or is at risk of developing anemia or blood loss. In another aspect, the invention relates to methods of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing a disease or condition associated with low red blood cell levels (e.g., low hemoglobin level, low red blood cell count, or low hematocrit, e.g., low red blood cell mass) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments, the disease or condition is anemia or blood loss.

[0044] In another aspect, the invention relates to a method of treating a subject having or at risk of developing anemia, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0045] In some embodiments of any of the above aspects, the anemia or blood loss is due to cancer, cancer treatment, kidney disease or renal failure (e.g., chronic kidney disease or acute kidney disease or failure), myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse reaction to a drug, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, liver disease (e.g., acute liver disease or chronic liver disease), diabetes, bleeding (e.g., acute or chronic bleeding), infection, hemoglobinopathies, drug use, alcohol abuse, elderly, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura Schoenlein-Henoch, Shwachman syndrome (e.g., Shwachman-Diamond syndrome), contraindications to blood transfusion, associated with surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0046] In some embodiments of any of the above aspects, the anemia is caused by chronic kidney disease. In some embodiments of any of the above aspects, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0047] In some embodiments of any of the above aspects, the subject does not respond well to treatment with erythropoietin (EPO) or is sensitive to the side effects of EPO. In some embodiments of any of the above aspects, the method increases erythropoiesis, red blood cell count, hematocrit, or hemoglobin levels (eg, red blood cell mass).

[0048] In some embodiments of any of the above aspects, the method reduces the subject's need for blood transfusions. In another aspect, the present invention relates to a method of preventing pulmonary hypertension (PH) in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0049] In another aspect, the present invention relates to a method of reducing the risk of developing PH in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0050] In another aspect, the present invention relates to a method of slowing or inhibiting the progression of PH in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0051] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0052] In another aspect, the invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0053] In another aspect, the present invention relates to a method of reducing vascular remodeling in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0054] In another aspect, the invention relates to a method of reducing right ventricular hypertrophy in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0055] In another aspect, the present invention relates to a method of decreasing pulmonary vascular resistance in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0056] In some embodiments of any of the above aspects, the PH is pulmonary arterial hypertension (PAH). In some embodiments, the PAH is idiopathic PAH. In some embodiments, the PAH is hereditary PAH. In some embodiments, the PAH is associated with HIV infection, schistosomiasis, liver cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., cocaine or methamphetamine use).

[0057] In some embodiments of any of the above aspects, the PH is venous PH. In some embodiments, the PH is associated with left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital or acquired pulmonary vein stenosis.

[0058] In some embodiments of any of the above aspects, the PH is hypoxic PH. In some embodiments, the hypoxic PH is associated with chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoventilation, chronic exposure to high altitude, or developmental abnormalities.

[0059] In some embodiments of any of the above aspects, the PH is thromboembolic PH. In some embodiments, the thromboembolic PH is associated with chronic thromboembolic pulmonary hypertension, pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection.

[0060] In some embodiments of any of the above aspects, the PH is other PH. In some embodiments, the other PH is associated with a blood disease (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0061] In some embodiments of any of the above aspects, the method reduces the frequency or severity of one or more symptoms of PH (e.g., reduces the severity or frequency of shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or pressure, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness, or fainting).

[0062] In some embodiments of any of the above aspects, the method reduces pulmonary vascular remodeling. In some embodiments of any of the above aspects, the method reduces vascular remodeling in the heart.

[0063] In some embodiments of any of the above aspects, the method reduces right ventricular hypertrophy. In some embodiments of any of the above aspects, the method reduces pulmonary vascular resistance (eg, reduces pulmonary vascular resistance compared to a measurement taken before treatment).

[0064] In some embodiments of any of the above aspects, the method improves performance in a 6-minute walk test (eg, improves performance compared to a measurement taken before treatment).

[0065] In some embodiments of any of the above aspects, the method reduces or inhibits binding of activin and / or myostatin to their endogenous receptors. In some embodiments of any of the aforementioned aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to reduce fibrosis, prevent the onset of fibrosis, delay or attenuate the onset of fibrosis, slow, inhibit, or reverse the progression of fibrosis, reduce the risk of developing fibrosis, reverse fibrosis, reduce one or more symptoms of fibrosis, improve the function of fibrotic tissue or organ, affect myostatin, activin, and / or BMP9 signaling in a subject, or inhibit the binding of activin and / or myostatin to their endogenous receptors.

[0066] In some embodiments of any of the foregoing aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to increase red blood cell levels, increase hemoglobin levels, increase hematocrit, increase red blood cell formation, increase red blood cell count, increase red blood cell mass, reduce the need for transfusions, treat anemia, affect myostatin, activin and / or BMP9 signaling in the subject, or reduce or inhibit binding of activin and / or myostatin to their endogenous receptors.

[0067] In some embodiments of any of the foregoing aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to prevent PH, reduce the risk of developing PH, reduce the severity or frequency of one or more symptoms of PH, delay or attenuate the onset of PH, delay or inhibit the progression of PH, treat PH, reduce pulmonary vascular remodeling, reduce cardiac vascular remodeling, reduce right ventricular hypertrophy, reduce pulmonary vascular resistance, improve performance in a 6-minute walk test, affect signaling of at least one of myostatin, activin, and BMP9 in a subject, or reduce or inhibit binding of activin and / or myostatin to their endogenous receptors. In some embodiments, the PH is PAH. In some embodiments, the PH is venous PH. In some embodiments, the PH is hypoxic PH. In some embodiments, the PH is thromboembolic PH. In some embodiments, the PH is other PH.

[0068] In some embodiments of any of the above aspects, the method does not cause a vascular complication in the subject. In some embodiments, the method does not increase vascular permeability or leakage.

[0069] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 69. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 58. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 6. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 38. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 41. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 44. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 70. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 71. In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 72. In some embodiments of any of the above aspects, the variant having the sequence of SEQ ID NO: 69, SEQ ID NO: 58, SEQ ID NO: 6, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72 is selected from the group consisting of X 17 In some embodiments of any of the above aspects, the variant having the sequence of SEQ ID NO:69, SEQ ID NO:58, SEQ ID NO:6, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72 has an amino acid K at position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the variant having the sequence of SEQ ID NO:69, SEQ ID NO:58, SEQ ID NO:6, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:70, SEQ ID NO:71 has 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 variant having the sequence of SEQ ID NO:69, SEQ ID NO:58, SEQ ID NO:6, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72, optionally at position X 17Amino acid K, position X 23 , X 24 , X 25 , and X 26 and / or a pharmaceutical composition containing the same, to increase, reduce, or prevent fibrosis in a subject in need thereof (e.g., a subject having fibrosis or at risk of developing fibrosis), slow or inhibit the progression of fibrosis in a subject in need thereof, reverse fibrosis in a subject in need thereof, treat a subject having fibrosis or at risk of developing fibrosis, increase myostatin, activin, and / or cytochrome P450 receptor activator (C-terminal extension) in a subject (e.g., a subject having or at risk of developing fibrosis, low red blood cell levels, or PH), or or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors), increasing red blood cell levels in a subject (e.g., a subject having or at risk of developing anemia or blood loss), increasing red blood cell formation in a subject (e.g., a subject having or at risk of developing anemia or blood loss), treating a subject having anemia or at risk of developing PH, preventing PH, reducing the risk of developing PH, slowing or inhibiting the progression of PH, or treating a subject having or at risk of developing PH.

[0070] definition As used herein, the term "extracellular activin type IIA receptor (ActRIIA) mutant" refers to a soluble extracellular portion of the single-pass transmembrane receptor ActRIIA having at least one amino acid substitution relative to wild-type extracellular ActRIIA (e.g., the bolded portion of the sequence of SEQ ID NO: 75 shown below), or a peptide containing extracellular ActRIIA having any one of the sequences of SEQ ID NOs: 76 to 96. The sequence of the wild-type human ActRIIA precursor protein is shown below (SEQ ID NO: 75), with the signal peptide in italics and the extracellular portion in bold.

[0071] Wild-type human ActRIIA precursor protein (SEQ ID NO: 75):

[0072] [ka]

[0073] The extracellular ActRIIA variant may have the sequence of any one of SEQ ID NOs: 1-72. In certain embodiments, the extracellular ActRIIA variant has the sequence of any one of SEQ ID NOs: 6-72 (Table 2). In some embodiments, the extracellular ActRIIA variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73).

[0074] As used herein, the term "extracellular ActRIIB mutant" refers to a peptide comprising a soluble extracellular portion of the single-pass 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 mutant may have the sequence of SEQ ID NO: 149, as shown below: Extracellular ActRIIB mutant (SEQ ID NO: 149):

[0075] [ka]

[0076] As used herein, the term "linker" refers to a bond between two elements, e.g., peptide or protein domains. The polypeptides described herein may include an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety. This moiety can increase the stability or improve the pharmacokinetic properties of the polypeptide. This moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide via a linker. The linker can be a covalent bond or a spacer. The term "bond" refers to any type of bond created by a chemical bond, e.g., an amide bond or a disulfide bond, or a chemical reaction, e.g., chemical conjugation. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids) that exists between two elements, e.g., peptide or protein domains, to provide space and / or flexibility between the two elements. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to a peptide spaced apart by the polypeptide backbone). For example, the formation of a disulfide bond between two hinge regions that form an Fc domain is not considered a linker.

[0077] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain comprises at least C H 2 domain and C H The Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) with a human Fc domain comprising three domains. The Fc domain monomer comprises a second and a third antibody constant domain (C H 2 and C HIn some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not include any portion of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are joined by two C H Dimers are formed through interactions between the three antibody constant domains and one or more disulfide bonds formed between the hinge domains of two dimerizing Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack effector function, exemplified by a "dead Fc domain." In certain embodiments, each Fc domain monomer of the Fc domain is mutated to reduce interaction or binding between the Fc domain and Fcγ receptors. H The Fc domain comprises an amino acid substitution within the antibody constant domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. The Fc domain may be of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, for example, a recombinant Fc domain.

[0078] As used herein, the term "albumin-binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and functions to bind to serum albumin. Albumin-binding peptides may be of different origins, e.g., human, mouse, or rat. In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0079] As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to transmembrane receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID NO:P02751 (SEQ ID NO:153). In other embodiments, the fibronectin domain is an Adnectin protein.

[0080] As used herein, the term "human serum albumin" refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood. Human serum albumin accounts for approximately half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0081] As used herein, the term "fused" is used to refer to the combination or joining of two or more elements, components, or protein domains, e.g., peptides or polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., amide bonds. For example, two single peptides can be fused in tandem via chemical conjugation, chemical bonds, peptide linkers, or any other covalent bonding means to form one continuous protein structure, e.g., a polypeptide. In some embodiments of the polypeptides described herein, an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) may be fused in tandem via a linker to the N-terminus or C-terminus of a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., the sequence of SEQ ID NO: 151), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide (e.g., the sequence of SEQ ID NO: 152), a fibronectin domain (e.g., the sequence of SEQ ID NO: 153), or human serum albumin (e.g., the sequence of SEQ ID NO: 154)). For example, an extracellular ActRIIA 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 dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a peptide linker, in which the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIA variant via a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a chemical bond, e.g., a peptide bond.

[0082] As used herein, the term "C-terminal extension" refers to the addition of one or more amino acids to the C-terminus of a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-70 (e.g., SEQ ID NOS: 6-70)). The C-terminal extension can be 1 to 6 amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids). Exemplary C-terminal extensions are the amino acid sequence NP (a 2-amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 155) (a 6-amino acid C-terminal extension). Any amino acid sequence that does not disrupt the activity of the polypeptide can be used. SEQ ID NO: 71, i.e., the sequence of SEQ ID NO: 69 with the C-terminal extension of NP, and SEQ ID NO: 72, i.e., the sequence of SEQ ID NO: 69 with the C-terminal extension of NPVTPK, represent two of the possible ways in which the polypeptides of the present invention can be modified to include a C-terminal extension.

[0083] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence, e.g., an extracellular ActRIIA variant, that are identical to the amino acid (or nucleic acid) residues in a reference sequence, e.g., a wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73), after aligning the sequences and introducing gaps as necessary to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways within the skill of the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for assessing alignment, including algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (alternatively, this can be expressed as a given candidate sequence having or containing a particular percent amino acid (or nucleic acid) sequence identity to a given reference sequence) is calculated as follows: 100 x (fraction A / B) where A is the number of amino acid (or nucleic acid) residues assigned an identity score in the alignment of the candidate sequence with the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments, if the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence will not be equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0084] In certain embodiments, a reference sequence aligned for comparison to a candidate sequence may show 50% to 100% identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. If a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0085] As used herein, the term "serum half-life," in the context of administering a therapeutic protein to a subject, refers to the time required for the plasma concentration of the protein to decrease by half in the subject. Proteins may be redistributed or eliminated from the bloodstream, or may be degraded, for example, by proteolysis. As described herein, a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) exhibits a serum half-life of 7 days in humans.

[0086] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner, for example, an extracellular ActRIIA mutant and BMP9 or activin A. Unless otherwise indicated, binding affinity refers to the intrinsic binding affinity, which represents a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally determined by the dissociation constant (K D ) or affinity constant (K A ) Two molecules that have low binding affinity for each other generally tend to bind slowly and dissociate easily, with a large K D Two molecules that have a high affinity for each other generally bind more easily and tend to remain bound longer, exhibiting a small K D The K of two interacting molecules is shown.D can be determined using methods and techniques well known in the art, such as, for example, surface plasmon resonance. D is k off / k on It is calculated as the ratio of

[0087] As used herein, the phrase "affecting myostatin, activin, and / or BMP9 signaling" means altering the binding of myostatin, activin, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA). In some embodiments, a polypeptide comprising an extracellular ActRIIA variant described herein reduces or inhibits the binding of myostatin, activin, and / or BMP9 to their receptors (e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA)). As described herein, a polypeptide of the present invention comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the 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 greater). D ).

[0088] As used herein, the terms "increase" and "decrease" refer to modulating, resulting in a greater or lesser amount of function, expression, or activity of a metric, respectively, compared to a reference. For example, following administration of a polypeptide of the invention comprising an extracellular ActRIIA variant in the methods described herein, the amount of a marker for a metric described herein (e.g., red blood cell count) may increase or decrease in a subject compared to the amount of the marker before administration. Generally, the metric is measured at least one week, one month, three months, or six months after administration, when administration has shown the recited effect, e.g., after the treatment regimen has begun.

[0089] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell, e.g., a human hair cell).

[0090] As used herein, the term "fibrosis" refers to a pathological process in which excessive fibrous connective tissue is formed. Fibrosis is characterized by the accumulation of fibroblasts and the deposition of collagen in excess of normal levels in a particular tissue. In response to inflammation or tissue injury, nearby fibroblasts migrate into the wound, proliferate, and produce large amounts of collagenous extracellular matrix. When fibrosis occurs in response to injury, the term "scarring" can be used synonymously. Fibrosis can occur in many tissues of the body, including, for example, the lung, skin, liver, kidney, heart, eye, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testes, ovaries, adrenal glands, arteries, veins, colon, small and large intestines, biliary tract, and intestines.

[0091] As used herein, the term "pulmonary hypertension" or "PH" refers to a disorder characterized by elevated blood pressure between the heart and lungs, and may include elevated blood pressure in the pulmonary arteries (pulmonary arterial hypertension), pulmonary veins, or pulmonary capillaries. Pulmonary hypertension is associated with many symptoms, including shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (e.g., ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or palpitations, bluish discoloration of the lips or skin (cyanosis), dizziness, or fainting. PH also reduces exercise tolerance and can lead to heart failure.

[0092] As used herein, the term "pulmonary arterial hypertension" or "PAH" refers to a form of pulmonary hypertension characterized by narrowing or obstruction of small pulmonary arteries, often caused by scarring, and elevated pulmonary artery blood pressure. PAH is also known as WHO Group I pulmonary hypertension. PAH can be diagnosed based on a resting mean pulmonary artery pressure greater than 25 mmHg with a normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, syncope, and other symptoms, all of which worsen with exertion. PAH can be a severe disease with markedly reduced exercise tolerance and heart failure. The two main types of PAH are idiopathic PAH (e.g., PAH without an identified predisposing factor) and hereditary PAH (e.g., PAH associated with mutations in BMPR2, ALK1, SMAD9, caveolin-1, KCNK3, or EIF2AK4). In 70% of familial PAH cases, mutations are located in the BMPR2 gene. Risk factors for developing PAH include a family history of PAH, drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), liver cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus).

[0093] As used herein, the terms "venous pulmonary hypertension" and "venous PH" refer to forms of pulmonary hypertension secondary to left heart disease. Venous PH is also known as WHO Group II PH. Venous PH can be associated with or caused by left ventricular systolic dysfunction (e.g., left ventricular failure), left ventricular diastolic dysfunction, valvular heart disease (e.g., mitral valve disease or aortic valve disease), congenital cardiomyopathy, or congenital or acquired pulmonary vein stenosis.

[0094] As used herein, the terms "hypoxic pulmonary hypertension" and "hypoxic PH" refer to a form of pulmonary hypertension resulting from lung disease or chronic hypoxia. This form of PH is also known as WHO Group III PH. Hypoxic PH can be associated with or caused by chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, chronic exposure to high altitude, or developmental abnormalities.

[0095] As used herein, the terms "thromboembolic pulmonary hypertension" and "thromboembolic PH" refer to a form of pulmonary hypertension associated with chronic arterial obstruction (e.g., a blood clot). Thromboembolic PH is also known as WHO Group IV PH. Thromboembolic PH can be associated with or caused by chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection).

[0096] As used herein, the terms "miscellaneous pulmonary hypertension" and "other PH" refer to forms of pulmonary hypertension with an unknown or multifactorial mechanism. This form of PH is classified as WHO Group V PH. Other PH is associated with hematological disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic diseases (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0097] As used herein, the term "red blood cell levels" refers to clinically observable metrics such as hematocrit, red blood cell count, and hemoglobin measurements. As used herein, the terms "increasing red blood cell levels" and "promoting erythropoiesis" refer to an increase in clinically observable metrics such as hematocrit, red blood cell count, and hemoglobin measurements, and are intended to be neutral with respect to the mechanism by which such changes occur. As used herein, the term "low red blood cell levels" refers to red blood cell count, hematocrit, and hemoglobin measurements that are below the range of values ​​considered normal for a subject's age and sex.

[0098] As used herein, the term "red blood cell mass" refers to the total mass of red blood cells in the circulation, which is typically reduced in anemia. As used herein, the terms "red blood cell formation" and "red cell production" refer to the production of red blood cells, such as the process of erythropoiesis in which red blood cells are produced in the bone marrow.

[0099] As used herein, the term "anemia" refers to an abnormality in hemoglobin or red blood cells that results in a decrease in the oxygen level in the blood.Anemia may be related to abnormalities in the production, processing, or function of red blood cells and / or hemoglobin.The term anemia refers to any decrease in the number of red blood cells and / or hemoglobin levels in the blood compared to normal blood levels.

[0100] As used herein, the term "vascular complications" refers to vascular disorders or any damage to blood vessels, for example, damage to the vascular wall. Damage to the vascular wall can lead to increased vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the vascular wall to allow small molecules, proteins, and cells to enter and exit the blood vessel. Increased vascular permeability or leakage can occur due to an increase in the gaps between endothelial cells lining the vascular wall (e.g., an increase in the size and / or number of gaps) and / or thinning of the vascular wall.

[0101] As used herein, the term "polypeptide" refers to a single polymer in which the monomers are amino acid residues covalently linked to one another through amide bonds. Polypeptide is intended to encompass any amino acid sequence that is naturally occurring, recombinant, or synthetically produced.

[0102] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. These two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, an Fc domain can be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein comprising an extracellular ActRIIA variant fused to an Fc domain monomer can form a homodimer through the interaction of the two Fc domain monomers, which form the Fc domain in the homodimer.

[0103] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. These two monomers can form a heterodimer through 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 through the interaction of two Fc domain monomers, each fused to a different ActRIIA variant, to form an Fc domain in the heterodimer.

[0104] As used herein, the term "host cell" refers to a vehicle containing necessary cellular components, e.g., organelles required for expressing a protein from a corresponding nucleic acid. The nucleic acid is generally contained within a nucleic acid vector, which can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell can be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or HEK293 cell).

[0105] As used herein, the term "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid, or vector of the invention, or a pharmaceutical composition containing a polypeptide, nucleic acid, or vector of the invention, that is effective to achieve a desired therapeutic effect in treating a patient with a disease or condition such as anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, other PH). In particular, a therapeutically effective amount of a polypeptide, nucleic acid, or vector avoids adverse side effects.

[0106] As used herein, the term "pharmaceutical composition" refers to a medicament or pharmaceutical formulation containing an active ingredient and excipients and diluents that can make the active ingredient suitable for the administration method. The pharmaceutical composition of the present invention contains pharmaceutically acceptable ingredients that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in the form of a tablet or capsule for oral administration, or in the form of an aqueous dosage form for intravenous or subcutaneous administration.

[0107] 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 deleterious 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 will vary depending on the mode of administration. For example, aqueous carriers are commonly used for intravenous administration, while solid carriers are preferred for oral administration.

[0108] As used herein, the term "treating and / or preventing" refers to the treatment and / or prevention of a disease or condition, e.g., anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) using the methods and compositions of the present invention. Generally, treatment of anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) occurs after a subject has developed and / or been diagnosed with the anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). Prevention of anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) refers to steps or procedures taken when a subject is at risk of developing that anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). The subject exhibits signs or mild symptoms that a physician determines to be an indication or risk factor for developing anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH); has another disease or condition associated with the development of anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH); is undergoing treatment that may cause anemia or fibrosis (e.g., surgery, chemotherapy, or radiation); or has a family history or genetic predisposition to developing anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) but has not yet developed such a disease or condition.

[0109] As used herein, the term "subject" refers to a mammal, such as, preferably, a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., cynomolgus monkeys), mice, dogs, cats, horses, and cows. [Effects of the Invention]

[0110] According to the present invention, activin type IIA receptor mutants and methods for using them are provided. [Brief explanation of the drawings]

[0111] [Figure 1-1] 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIA and ActRIIB and the amino acid substitutions of ActRIIA mutants. [Figure 1-2] 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIA and ActRIIB and the amino acid substitutions of ActRIIA mutants. [Figure 2] 1 is a series of graphs showing that treatment of aged mdx mice with ActRIIA / B-Fc (A / B, SEQ ID NO: 69 fused to the Fc domain) attenuated the development of fibrosis. [Figure 3] 1 is a graph showing that treatment of non-human primates with ActRIIA / B-Fc (SEQ ID NO: 69 fused to the Fc domain) increased red blood cell mass. Hct - hematocrit, Hgb - hemoglobin, RBC - red blood cell count. DETAILED DESCRIPTION OF THE INVENTION

[0112] The present invention relates to polypeptides comprising extracellular activin type IIA receptor (ActRIIA) variants. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIA variant fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIA variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interaction between two Fc domain monomers. The ActRIIA variants described herein have weaker or no binding affinity for bone morphogenetic protein 9 (BMP9) compared to activin and myostatin. The present invention also includes methods for treating or preventing fibrosis, treating or preventing low red blood cell levels (such as anemia or blood loss) by increasing red blood cell levels (e.g., by increasing red blood cell count, hemoglobin levels, or hematocrit, e.g., red blood cell mass) or red blood cell production, treating or preventing pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), 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.

[0113] I. Extracellular activin type IIA receptor (ActRIIA) mutants The activin type II receptor is a single-transmembrane domain receptor that mediates signaling for ligands in the transforming growth factor β (TGF-β) superfamily. TGF-β superfamily ligands are involved in many physiological processes in the host, including muscle growth, vascular growth, cell differentiation, homeostasis, and bone formation. Examples of TGF-β superfamily ligands include activin, inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin, and GDF11), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activin have been shown to regulate fibrosis. For example, mice lacking myostatin exhibit reduced muscle fibrosis, and injection of myostatin-coated beads induces muscle fibrosis in mice. Mice overexpressing an activin subunit that induces the production of diffusible activin A also exhibit fibrosis. Elevated activin A has also been observed in clinical and experimental pulmonary hypertension. Another activin type II receptor ligand, GDF11, has been shown to be overexpressed in mouse models of β-thalassemia and associated with ineffective red blood cell production. Therefore, methods to reduce or inhibit signaling through activin type II receptors (e.g., signaling mediated by myostatin, activin, and / or GDF11) could be used to treat fibrosis, diseases associated with reduced red blood cell levels (e.g., anemia), and pulmonary hypertension (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, and other PH).

[0114] There are two types of activin type II receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds to ActRIIB with approximately 300-fold higher binding affinity than ActRIIA (see, for example, Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIA is known to have a longer half-life than ActRIIB. The present invention describes extracellular ActRIIA variants constructed by introducing amino acid residues from ActRIIB into ActRIIA with the goal of conferring the physiological properties conferred by ActRIIB while also maintaining the beneficial physiological and pharmacokinetic properties of ActRIIA. An optimal peptide, for example, reduces fibrosis, treats PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, and other PH), and / or increases red blood cell levels (e.g., increases red blood cell production), while maintaining a longer serum half-life and low binding affinity for BMP9. Preferred ActRIIA variants also exhibit improved binding to activin and / or myostatin compared to wild-type ActRIIA, allowing them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling. These variants can be used to treat disorders in which activin receptor signaling is elevated, such as fibrosis, PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), and anemia, resulting in reduced fibrosis (e.g., reduced fibrosis or slowed or stopped the progression of fibrosis, improved existing fibrosis, or reversed existing fibrosis), increased red blood cell levels (e.g., increased hemoglobin level, hematocrit, or red blood cell count, e.g., increased red blood cell production and / or red blood cell mass), or reduced symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, amino acid substitutions can be introduced into extracellular ActRIIA mutants to reduce or eliminate the binding affinity of the mutants for BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIA and ActRIIB are shown below.

[0115] Human ActRIIA extracellular portion (SEQ ID NO: 73):

[0116] [ka]

[0117] Human ActRIIB extracellular portion (SEQ ID NO: 74):

[0118] [ka]

[0119] The polypeptides described herein include extracellular ActRIIA mutants having at least one amino acid substitution relative to a wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73 or an extracellular ActRIIA having any one of the sequences of SEQ ID NOs: 76-96. Potential amino acid substitutions at 27 different positions can be introduced into the extracellular ActRIIA mutants (Table 1). In some embodiments, the extracellular ActRIIA mutants can have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73). The extracellular ActRIIA variant may have one or more (e.g., 1-27, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) amino acid substitutions relative to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73). In some embodiments, the extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of SEQ ID NO: 1) may contain amino acid substitutions at all 27 positions as listed in Table 1. In some embodiments, the extracellular ActRIIA variant may contain amino acid substitutions at multiple positions, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, of the 27 positions listed in Table 1.

[0120] Amino acid substitutions can decrease or increase the activity and / or binding affinity of the ActRIIA variants of the present invention. To maintain polypeptide function, it is important to retain the lysine (K) at position X17 of the sequences shown in Tables 1 and 2 (SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)). Substitutions at that position can result in loss of activity. For example, An ActRIIA mutant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reduced activity in vivo, 17 This indicates that substitution of lysine (K) with alanine (A) at position X is not tolerated. Thus, the variants in Tables 1 and 2 (e.g., ActRIIA variants of the present invention including SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) have a lysine (K) at position X 17 It holds the amino acid K.

[0121] The ActRIIA variants of the present invention preferably have reduced, weak, or substantially no binding to BMP9. 23 , X 24 , X 25 , and X 26 ActRIIA variants containing the amino acid sequence TEEN at position X 24 Maintaining amino acid K at position X 23 , X 24 , X 25 , and X 26 A mutant having the amino acid sequence TKEN at 1 has reduced BMP9 binding (e.g., reduced compared to wild-type ActRIIA). The sequences TEEN and TKEN can be used interchangeably to provide reduced BMP9 binding in ActRIIA mutants of the present invention (e.g., mutants in Tables 1 and 2, e.g., SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)).

[0122] ActRIIA variants of the present invention may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). A C-terminal extension can include the addition of 1 to 6 additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus of any of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). One potential C-terminal extension that can be included in an ActRIIA variant of the present invention is the amino acid sequence NP. For example, a sequence including the C-terminal extension NP is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). Another exemplary C-terminal extension that can be included in an ActRIIA variant of the present invention is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, a sequence including the C-terminal extension NPVTPK is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK).

[0123] [Table 1]

[0124] In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 2, X3 is E, X6 is R, and X 11 is D and X 12 is K and X 13 is R and X 16 is K or R, and X 17 is K and X 19 is W and X 20 is L and X 21 is D and X 22 is I or F. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X 17 is K. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1-3, X 17 is K and X 23 is T and X 24 is E and X 25 is E and X 26is N. In some embodiments of an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1 to 5, X 17 is K and X 23 is T and X 24 is K and X 25 is E and X 26 is N.

[0125] In some embodiments, the polypeptides described herein comprise an extracellular ActRIIA mutant having the sequence of any one of SEQ ID NOs: 6-72 (Table 2).

[0126] [Table 2-1]

[0127] [Table 2-2]

[0128] [Table 2-3]

[0129] [Table 2-4]

[0130] In some embodiments, the polypeptide of the present invention comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) has a sequence at position X 17 It has amino acid K at position X. 17Altering the amino acids in the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) results in reduced activity. 17 This indicates that substitution of K with A in

[0131] In some embodiments, position X 23 , X 24 , X 25 , and X 26 The polypeptide of the present invention, which comprises an extracellular ActRIIA variant (e.g., any one of SEQ ID NOS: 1 to 72 (e.g., SEQ ID NOS: 6 to 72)) having the sequence TEEN at position X 24 In some embodiments, at position X, the amino acid E may be substituted with the amino acid K. 23 , X 24 , X 25 , and X 26 The polypeptide of the present invention, which comprises an extracellular ActRIIA mutant (e.g., any one of SEQ ID NOS: 1 to 72 (e.g., SEQ ID NOS: 6 to 72)) having the sequence TKEN at position X 24 At position X, the amino acid K may be substituted with the amino acid E. 23 , X 24 , X 25 , and X 26 Polypeptides having the sequence TEEN or TKEN at position 1 have reduced or weak binding to BMP9 (e.g., reduced binding to BMP9 compared to the BMP9 binding of wild-type ActRIIA).

[0132] 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, a sequence comprising the C-terminal extension NP 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, a sequence comprising the C-terminal extension NPVTPK is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK). The C-terminal extension can include the addition of 1 to 6 additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus.

[0133] In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIA variant may further comprise a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin), which can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIA variant via a linker or other covalent bond. A polypeptide comprising an extracellular ActRIIA variant fused to an Fc domain monomer can form a dimer (e.g., a homodimer or heterodimer) through interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.

[0134] In some embodiments, the extracellular ActRIIA mutant described herein does not have any of the sequences of SEQ ID NOs: 76-96 shown in Table 3 below.

[0135] [Table 3-1]

[0136] [Table 3-2]

[0137] Furthermore, in some embodiments, the polypeptides described herein have a serum half-life in humans of at least 7 days. D The polypeptide can bind to bone morphogenetic protein 9 (BMP9) at a K of 10 pM or greater. D In some embodiments, the polypeptide may bind to activin A at position X. In some embodiments, the polypeptide does not bind to BMP9 or activin A. In some embodiments, the polypeptide binds to activin and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., reduced BMP9 binding compared to BMP9 binding of wild-type ActRIIA). In some embodiments, the polypeptide with low or weak binding to BMP9 has a structure at position X. 23 , X 24 , X 25 , and X 26 has the sequence TEEN or TKEN.

[0138] Additionally, in some embodiments, the polypeptide has a K of about 200 pM or greater. D (e.g., K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or greater) D , e.g., a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or greater D , e.g., a K between about 200 pM and about 50 nM D In some embodiments, the polypeptide does not substantially bind to human BMP9. In some embodiments, the polypeptide has 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, e.g., K between about 800 pM and about 200 pM D In some embodiments, the polypeptide may bind to human activin A with a K of 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , e.g., K between about 800 pM and about 200 pM D ) can bind to human activin B. The polypeptide has a K of approximately 5 pM or greater. D (e.g., a K of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or more) D ) and can also bind to growth and differentiation factor 11 (GDF-11).

[0139] II. Fc Domain In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to extend the serum half-life of the polypeptide. A polypeptide comprising an extracellular ActRIIA variant fused to an Fc domain monomer can form a dimer (e.g., a homodimer or a heterodimer) through interaction between two Fc domain monomers, which form an Fc domain in the dimer. As conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain is a C-terminal fragment of an immunoglobulin. HThe Fc domain comprises two Fc domain monomers that form a dimer through interaction between three antibody constant domains. A wild-type Fc domain forms the minimal structure required for binding to an Fc receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, or FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, typically resulting in a "dead" Fc domain. For example, the Fc domain may contain specific amino acid substitutions known to minimize interaction between the Fc domain and an Fcγ receptor. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Kabat numbering of amino acid residues can be determined for a given antibody by aligning homologous regions of the antibody sequence with the "standard" Kabat numbering sequence. Furthermore, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a polypeptide dimer comprising an extracellular ActRIIA variant fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. The sequence of an Fc domain monomer that can be fused to an extracellular ActRIIA variant is shown below (SEQ ID NO: 97):

[0140] [ka]

[0141] In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO: 97. In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO: 97. In some embodiments, an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97) by conventional genetic or chemical means, for example, 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.

[0142] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to an Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. In some embodiments, the Fc domain comprises a hinge domain. The Fc domain may be of the immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, for example, a recombinant Fc domain.

[0143] Methods for creating Fc domains with reduced dimerization are known in the art. In some embodiments, C is substituted with C to impair dimerization through steric clashes. H 3-C H One or more amino acids with large side chains (e.g., tyrosine or tryptophan) may be introduced into the C3 dimer interface to remove favorable interactions.H 3-C H 3 One or more amino acids with small side chains (e.g., alanine, valine, or threonine) may be introduced into the dimer interface. H Methods for introducing amino acids with large or small side chains into the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patent No. 8,216,805 and U.S. Patent No. 5,731,168, Ridgway et al. (Protein Eng. 9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26-35, 1997), and Merchant et al. (Nat Biotechnol. 16:677-681, 1998), all of which are incorporated herein by reference in their entirety.

[0144] In yet another embodiment, a C between two Fc domains H 3-C H 3C that constitutes the interface H One or more amino acid residues in the three domains are substituted with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) so that the interaction is electrostatically unfavorable due to the introduced amino acid of the particular charge. H Methods for introducing charged amino acids into the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patent Application Publication No. 2012 / 0244578, and U.S. Patent Application Publication No. 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0145] In some embodiments of the invention, the Fc domain contains 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 set forth in SEQ ID NO: 151.

[0146] III. Albumin-binding peptides In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to a serum protein-binding peptide. Binding to the serum protein peptide can improve the pharmacokinetics of the protein pharmaceutical.

[0147] By way of example, albumin-binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin-binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0148] In the present invention, to extend the serum half-life of an extracellular ActRIIA variant, an albumin-binding peptide can be linked to the N-terminus or C-terminus (e.g., the C-terminus) of an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). In some embodiments, the albumin-binding peptide is linked to the N-terminus or C-terminus of the extracellular ActRIIA variant directly or via a linker.

[0149] In some embodiments, an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) can be fused to the N- or C-terminus of an albumin-binding peptide (e.g., SEQ ID NOS: 152) by conventional genetic or chemical means, for example, chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and the albumin-binding peptide. Without being bound by theory, it is believed that the inclusion of an albumin-binding peptide in the extracellular ActRIIA variant described herein may result in extended retention of the therapeutic protein via its binding to serum albumin.

[0150] IV. Fibronectin Domain In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to a fibronectin domain, which can improve the pharmacokinetics of the protein pharmaceutical.

[0151] A fibronectin domain is, for example, a high-molecular-weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, to extend the serum half-life of an extracellular ActRIIA mutant, a fibronectin domain is linked to the N-terminus or C-terminus (e.g., the C-terminus) of an extracellular ActRIIA mutant described herein (e.g., an extracellular ActRIIA mutant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). The fibronectin domain can be linked to the N-terminus or C-terminus of the extracellular ActRIIA mutant directly or via a linker.

[0152] By way of example, fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID NO:P02751 (SEQ ID NO:153). In another embodiment, the fibronectin domain is an Adnectin protein.

[0153] In some embodiments, an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) can be fused to the N- or C-terminus of a fibronectin domain (e.g., SEQ ID NOS: 153) by conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and the fibronectin domain. Without being bound by theory, it is believed that the inclusion of a fibronectin domain in the extracellular ActRIIA variant described herein may result in extended retention of the therapeutic protein through its binding to integrins and extracellular matrix components such as collagen and fibrin.

[0154] V. Serum Albumin In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of protein pharmaceuticals.

[0155] Serum albumin is a globular protein that is the most abundant blood protein in mammals. It is produced in the liver and accounts for approximately half of serum proteins. Serum albumin is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure required for proper distribution of body fluids between blood vessels and body tissues. In a preferred embodiment, the serum albumin is human serum albumin. In some embodiments of the present invention, to extend the serum half-life of an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)), human serum albumin is linked to the N-terminus or C-terminus (e.g., C-terminus) of the extracellular ActRIIA variant. Human serum albumin can be linked to the N-terminus or C-terminus of the extracellular ActRIIA variant directly or via a linker.

[0156] By way of example, serum albumin that can be used in the methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0157] In some embodiments, an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N-terminus or C-terminus of human serum albumin (e.g., SEQ ID NO: 154) by conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and human serum albumin. Without being bound by theory, it is believed that the inclusion of human serum albumin in the extracellular ActRIIA variant described herein may result in extended retention of the therapeutic protein.

[0158] VI. Linker The polypeptides described herein may include an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety via a linker. In some embodiments, the moiety increases the stability of the polypeptide. Examples of the moiety include an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin. In the present invention, the linker between a certain portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., SEQ ID NO: 151), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide (e.g., SEQ ID NO: 152), a fibronectin domain (e.g., SEQ ID NO: 153), or human serum albumin (e.g., SEQ ID NO: 154)) and an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) can be an amino acid spacer containing 1 to 200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the spacer may include a GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 98), GGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 100), GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), or SGGG (SEQ ID NO: 107) motif, e.g., multiple or repeated motifs.In some embodiments, the spacer can include 2 to 12 amino acids containing a GA or GS motif, e.g., GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer can include 3 to 12 amino acids containing a GGA or GGS motif, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). Further, in some embodiments, the spacer can comprise 4 to 12 amino acids including the motifs GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), e.g., GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In some embodiments, the spacer can comprise the motifs GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128) and GGGGSGGGGSGGGGGS (SEQ ID NO: 129).In some embodiments of the present invention, the amino acid spacer between a certain portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or serum albumin) and an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) may be GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

[0159] In some embodiments, the spacer may also include amino acids other than glycine, alanine, and serine, for example, AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146). In some embodiments, the spacer may include a motif, e.g., a multiple or repeated motif, of EAAAK (SEQ ID NO: 147). ... n (wherein X can be any amino acid (e.g., A, K, or E) and n is 1 to 5), and can include motifs, e.g., multiple or repeated motifs, of proline-rich sequences such as PAPAP (SEQ ID NO: 148).

[0160] The length of the peptide spacer and amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid the formation of aggregates.

[0161] VII. Vectors, Host Cells, and Protein Production The polypeptides of the present invention can be produced from host cells. A host cell refers to a vehicle containing necessary cellular components, e.g., organelles required for expressing the polypeptides and fusion polypeptides described herein from the corresponding nucleic acids. These nucleic acids may be contained within a nucleic acid vector, which can be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, or infection). The choice of nucleic acid vector will depend in part on the host cell used. Generally, preferred host cells are of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0162] Nucleic acid vector construction and host cells Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptides of the present invention can be obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type extracellular ActRIIA can be mutated to contain specific amino acid substitutions using standard techniques in the art, such as QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0163] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing the nucleic acid molecule in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may contain various components, which can be adjusted and optimized to suit a specific host cell. For example, vector components include, but are not limited to, a replication origin, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding a target protein, and a transcription termination sequence.

[0164] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F) cells, Chinese hamster ovary (CHO) cells, HeLa cells, COS cells, PC3 cells, Vero cells, MC3T3 cells, NS0 cells, Sp2 / 0 cells, VERY cells, BHK cells, MDCK cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, NS0 cells (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O cells, and HsS78Bst cells. In some embodiments, Escherichia coli (E. coli) cells can also be used as host cells of the present invention. E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. An appropriate cell line or host system can be selected to ensure proper modification and processing of the expressed polypeptide. The above-described expression vectors can be introduced into appropriate host cells using conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector has been introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify the gene encoding the desired sequence.Methods for expression of therapeutic proteins are known in the art, see, e.g., 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).

[0165] Protein Production, Recovery and Purification Host cells used to produce the polypeptides of the present invention can be grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) with any necessary supplementation of a selection agent, e.g., ampicillin. Host cells are cultured at a suitable temperature, e.g., about 20°C to about 39°C, e.g., 25°C to about 37°C, preferably 37°C, and at a CO2 level, e.g., 5-10%. The pH of the medium is generally about 6.8 to 7.4, e.g., 7.0, depending primarily on the host organism. When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter.

[0166] In some embodiments, depending on the expression vector and host cell used, the expressed protein can be secreted from the host cell (e.g., mammalian host cell) into the cell culture medium. Protein recovery can include filtering the cell culture medium to remove cellular debris. These proteins may be further purified. The polypeptides of the present invention can be purified by any method known in the art of protein purification, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Proteins can be isolated and purified, for example, by appropriately selecting an affinity column, such as a Protein A column (e.g., POROS Protein A chromatography), in combination with a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis.

[0167] In other embodiments, host cells can be disrupted, for example, by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, the cellular debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be linked to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His-tag), which binds with micromolar affinity to nickel-functionalized agarose affinity columns. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0168] Alternatively, the polypeptides of the present invention can be produced by cells of a subject (e.g., a human) by administering a vector (e.g., a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector, e.g., Modified Vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding a polypeptide of the present invention, e.g., in gene therapy. Once in the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), the vector promotes expression of the polypeptide, which is then secreted from the cells. If treatment of the disease or disorder is the desired outcome, no further action may be required. If collection of protein is desired, blood can be drawn from the subject and the protein can be purified from the blood by methods known in the art.

[0169] VIII. Pharmaceutical Compositions and Formulations The present invention relates to pharmaceutical compositions comprising a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72))). In some embodiments, the pharmaceutical compositions of the present invention comprise, as a therapeutic protein, a polypeptide comprising an extracellular ActRIIA variant having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1 to 70 (e.g., SEQ ID NOs: 6 to 70)). In some embodiments, the pharmaceutical compositions of the present invention comprise, as a therapeutic protein, a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin). In some embodiments, the pharmaceutical compositions of the present invention comprising the polypeptides of the present invention can be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions in therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers or excipients and can be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical compositions of the present invention comprise a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding a polypeptide of the present invention or a vector containing such a nucleic acid molecule.

[0170] Acceptable carriers and excipients for pharmaceutical compositions are non-toxic to recipients at the dosage and concentration used.Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; and carbohydrates such as glucose, mannose, sucrose, and sorbitol.The pharmaceutical composition of the present invention can be administered parenterally in the form of an injection preparation.Injectable pharmaceutical compositions can be prepared using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). Formulation methods are known in the art, see, for example, Banga (ed.), Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd Edition), Taylor & Francis Group, CRC Press (2015).

[0171] The pharmaceutical compositions of the present invention may be formulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the present invention may also be formulated in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and Practice of Pharmacy, 22nd Edition (2012). Pharmaceutical compositions used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0172] The pharmaceutical compositions of the present invention may also be prepared as sustained-release formulations. Suitable sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides of the present invention. Examples of sustained-release matrices include polyesters, hydrogels, polyactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT™), and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations enable release of molecules over several months, e.g., 1 to 6 months, while other formulations release the pharmaceutical compositions of the present invention over shorter periods, e.g., days to weeks.

[0173] The pharmaceutical composition can be prepared in a unit dosage form, if necessary. The amount of the active ingredient, e.g., the polypeptide of the present invention, contained in the pharmaceutical preparation is such that an appropriate dose is provided within the specified range (e.g., a dose within the range of 0.01 to 100 mg / kg body weight).

[0174] Pharmaceutical compositions for gene therapy may be in an acceptable diluent or may comprise a slow-release matrix in which the gene delivery vehicle is embedded. When hydrodynamic injection is used as the delivery method, pharmaceutical compositions containing nucleic acid molecules encoding the polypeptides described herein or vectors (e.g., viral vectors) containing the nucleic acid molecules are easily delivered intravenously in large liquid volumes. Vectors that can be used as in vivo gene delivery vehicles include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutant vaccinia Ankara), adeno-associated virus vectors, and alphavirus vectors.

[0175] IX. Route, Dosage, and Administration The pharmaceutical composition comprising the polypeptide of the present invention as therapeutic protein can be prepared for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration or intraperitoneal administration.The pharmaceutical composition can also be prepared for or administered via oral administration, nasal administration, spray administration, aerosol administration, rectal or vaginal administration.For injection preparations, various effective pharmaceutical carriers are known in the art.For example, see ASHP Handbook on Injectable Drugs, Toissel, 18th Edition (2014).

[0176] In some embodiments, pharmaceutical compositions comprising nucleic acid molecules encoding the polypeptides of the present invention or vectors containing such nucleic acid molecules can be delivered by gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutated vaccinia Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, mRNA molecules encoding the polypeptides of the present invention can be directly administered to a subject.

[0177] In some embodiments of the present invention, nucleic acid molecules encoding the polypeptides described herein or vectors containing such nucleic acid molecules can be administered using a hydrodynamic injection platform. In hydrodynamic injection, nucleic acid molecules encoding the polypeptides described herein are placed under the control of a strong promoter within an engineered plasmid (e.g., a viral plasmid). Plasmids are often easily delivered intravenously in large fluid volumes. Hydrodynamic injection uses controlled hydrodynamic pressure within a vein to increase cell permeability, so that the high pressure resulting from the rapid injection of a large fluid volume results in extravasation of the fluid and plasmid from the vein. Expression of nucleic acid molecules is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding the polypeptides described herein can be administered using hydrodynamic injection.

[0178] The dosage of the pharmaceutical composition of the present invention depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics, such as age, weight, and health status. The pharmaceutical composition of the present invention may contain a polypeptide of the present invention at a dose ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), or in more specific embodiments, about 0.1 to about 30 mg / kg, and even more specific embodiments, about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors, such as the extent of the subject's disease and various parameters.

[0179] Pharmaceutical compositions are administered in a manner appropriate for the dosage form and in a therapeutically effective amount to improve or remedy symptoms. Pharmaceutical compositions are administered in a variety of dosage forms, including intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Generally, therapeutic proteins are administered at 0.1-100 mg / kg, e.g., 1-50 mg / kg. Pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof, for example, daily, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, or one or more times (e.g., 1-10 times or more) as medically necessary. In some embodiments, pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof weekly, biweekly, monthly, bimonthly, or quarterly. Doses can be provided in single or multiple dosing regimens. The timing between doses can be decreased as the medical condition improves or increased as the patient's health declines.

[0180] X. Treatment method The present invention is based on the discovery that amino acid substitution from the extracellular portion of ActRIIB to the extracellular portion of ActRIIA results in ActRIIA variants with improved properties. ActRIIA variants created by introducing residues from ActRIIB into ActRIIA retain the beneficial properties of ActRIIA, such as longer serum half-life and lower binding affinity for BMP9, and acquire some of the beneficial properties of ActRIIB, such as enhanced binding to activin A and B (see Table 5 in Example 1). These ActRIIA variant properties create useful therapeutics that can compete with endogenous activin receptors for ligand binding. Because ActRIIA variants contain the extracellular portion of the receptor, they are soluble and can bind and sequester ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Thus, extracellular ActRIIA variants can be used to treat diseases or conditions associated with increased activin signaling (e.g., diseases or conditions associated with increased expression of activin receptors or activin receptor ligands). For example, loss of myostatin has been shown to reduce fibrosis, while increased expression of myostatin or activin induces fibrosis. In another example, the activin receptor ligand GDF11 is overexpressed in a mouse model of hemolytic anemia and is associated with defects in red blood cell production. In addition, activin A has also been found to be elevated in clinical and experimental pulmonary hypertension. Without wishing to be bound by theory, therapeutic agents that bind to activin receptor ligands (e.g., myostatin, activin, and / or GDF11) and reduce their interaction with endogenous activin receptors can be used to treat fibrosis, anemia, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).

[0181] The present invention provides compositions and therapeutic methods that can be used to reduce fibrosis, prevent fibrosis, reduce the risk of developing fibrosis, slow or stop the progression of fibrosis, or reverse fibrosis in a subject in need thereof. The present invention also provides compositions and therapeutic methods that can be used to increase red blood cell levels (e.g., increasing hemoglobin, increasing hematocrit, or increasing red blood cell count, e.g., increasing red blood cell production and / or red blood cell mass) in a subject in need thereof. The present invention further provides compositions and therapeutic methods that can be used to treat, reduce PH (e.g., reduce symptoms of PH such as shortness of breath (dyspnea), fatigue, swelling (e.g., edema) in the legs, feet, abdomen (e.g., ascites), or neck, chest pain or pressure, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness, or fainting), or slow or stop the progression of PH in a subject in need thereof. The present invention can treat, slow or stop the progression of any type of PH, such as PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.In some embodiments, the subject is diagnosed with a disease or condition associated with fibrosis (e.g., liver cirrhosis, pulmonary fibrosis, Crohn's disease, chronic kidney disease); a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss); a disease or condition associated with PH (e.g., a disease or condition associated with PAH, such as HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, liver cirrhosis, congenital heart abnormalities, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use)); a disease or condition associated with venous PH, such as left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; a disease or condition associated with chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., , sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), impaired alveolar hypoventilation, chronic exposure to high altitude, or developmental abnormalities; diseases or conditions associated with thromboembolic PH, such as chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, parasitic infection); other diseases or conditions associated with PH, such as blood diseases (e.g., chronic hemolytic anemia, sickle cell disease), systemic diseases (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension (pulmonary hypertension limited to one or more lobes of the lung). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling in a subject with a disease or condition comprising fibrosis, low red blood cell levels, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, other PH). In some embodiments, a polypeptide comprising an extracellular ActRIIA variant described herein reduces or inhibits binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors (e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA)).In some embodiments, affecting myostatin, activin, and / or BMP9 signaling (e.g., decreasing or inhibiting the binding of myostatin, activin, and / or BMP9 to their respective receptors, e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA)) can result in reduced fibrosis, reduced risk of developing fibrosis, delayed onset of fibrosis, reduced (e.g., slowed or inhibited) progression of fibrosis, reversal of fibrosis, increased red blood cell counts (e.g., increased erythrocyte counts), and increased erythrocyte counts (e.g., increased erythrocyte counts). For example, an increase in hemoglobin, hematocrit, or red blood cell count, e.g., an increase in red blood cell production and / or red blood cell mass), a reduction in symptoms of PH (e.g., a reduction in shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or heart palpitations, bluish discoloration of the lips or skin (cyanosis), dizziness, or fainting), a reduction in the risk of developing PH, a delay in the onset of PH, or a reduction in the progression of PH (e.g., slowing or preventing progression). PH may be PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.

[0182] The compositions and methods described herein can be used to treat and / or prevent (e.g., prevent the onset of or treat a subject diagnosed with) a medical condition, such as low red blood cell levels (e.g., low hemoglobin levels or low red blood cell count), fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)), e.g., an effective amount of an ActRIIA variant) can be administered to increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, increase red blood cell count, increase red blood cell mass, or increase red blood cell formation) in a subject in need thereof. The polypeptides described herein can increase red blood cell levels (e.g., increase hemoglobin levels, red blood cell count, hematocrit, red blood cell mass, or red blood cell formation) compared to measurements obtained before treatment. In some embodiments, the subject can have a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss). In some embodiments, the subject can have or be at risk of developing anemia or blood loss (e.g., the subject can be at risk of developing anemia due to other diseases or conditions, such as chronic kidney disease, rheumatoid arthritis, cancer, or inflammatory diseases (e.g., Crohn's disease, SLE, ulcerative colitis), or due to medical treatments, such as chemotherapy, radiation therapy, or surgery). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject having a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss).

[0183] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)), e.g., an effective amount of an ActRIIA variant) can be administered to prevent or reduce fibrosis in a subject in need thereof. In some embodiments, a polypeptide described herein can be administered to slow or halt the progression of fibrosis, reduce the risk of developing fibrosis, reduce one or more symptoms of fibrosis (e.g., reduce the frequency or severity), or reverse fibrosis. A polypeptide described herein can reduce fibrosis, or slow or reverse the progression of fibrosis, at least as compared to the progression of fibrosis before treatment or as compared to the progression of fibrosis in an untreated subject. In some embodiments, the subject may have or be at risk for developing fibrosis (e.g., the subject may have a disease or condition associated with fibrosis, such as a wound, hepatitis B or C, fatty liver disease, kidney disease (e.g., chronic kidney disease), heart disease, or atherosclerosis, or may be undergoing a treatment associated with the development of fibrosis, such as chemotherapy, radiation therapy, or surgery). In some embodiments, the polypeptides described herein prevent, delay, or attenuate the development of fibrosis in a subject at risk for developing fibrosis (e.g., the subject may be being treated with chemotherapy, radiation therapy, or surgery, or the subject may have a disease or condition associated with fibrosis, such as a wound, hepatitis B or C, fatty liver disease, kidney disease (e.g., chronic kidney disease), heart disease, or atherosclerosis). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject with fibrosis or a disease or condition associated with fibrosis.

[0184] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), e.g., an effective amount of an ActRIIA variant) can be administered to a subject in need thereof to treat PH, reduce PH (e.g., reduce the severity or frequency of one or more symptoms of PH, such as shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (e.g., ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or palpitations, bluish color of the lips or skin (cyanosis), dizziness or fainting), or prevent PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) (e.g., prevent the onset of PH). In some embodiments, the polypeptides described herein can be administered to slow or halt the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) or to reduce the risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). The polypeptides described herein can reduce the symptoms of PH (e.g., reduce the severity or frequency of one or more symptoms of PH, such as shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (e.g., ascites), neck (e.g., edema), chest pain or pressure, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness or fainting) compared to the symptoms or progression observed before treatment or compared to the symptoms or progression of PH in an untreated subject, or slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).In some embodiments, the subject may have PH or be at risk for developing PH (e.g., the subject may have idiopathic PAH); the subject may have a disease or condition associated with PAH (e.g., a condition that induces an increased risk of developing PAH), such as HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart abnormalities, connective tissue / autoimmune diseases (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use). The subject may have a disease or condition associated with venous PH (e.g., a disease or condition that increases the risk of developing venous PH), such as left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; the subject may have a chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoplasia, or other conditions associated with venous PH. The subject may have a disease or condition associated with hypoxic PH (e.g., a disease or condition that increases the risk of developing hypoxic PH), such as impaired ventilation, chronic exposure to high altitude, or a developmental abnormality; the subject may have a disease or condition associated with thromboembolic PH (e.g., a disease or condition that increases the risk of developing thromboembolic PH), such as chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection); or The subject may have other diseases or conditions associated with PH (e.g., diseases or conditions that confer an increased risk of developing other PH), such as a blood disorder (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disease (e.g., glycogen storage disease, Gaucher disease, thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.In some embodiments, the polypeptides described herein prevent, delay, or attenuate the onset of PH in a subject at risk of developing PH (e.g., the subject has a family history of PH (e.g., hereditary PAH), or the subject has a pre-existing condition known as PAH (e.g., HIV infection, schistosomiasis, cirrhosis of the liver, congenital heart defects, connective tissue / autoimmune diseases such as scleroderma and lupus), or drug use or abuse (e.g., methamphetamine or cocaine use), venous PH (e.g., left ventricular hypertrophy), or vasoconstriction (vasoconstriction). left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis), hypoxic PH (e.g., chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoventilation, chronic exposure to high altitude, or developmental abnormality), thromboembolic PH (e.g., chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection)), or Other PH (e.g., having a disease or condition that causes an increased risk of developing a blood disorder (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disease (e.g., glycogen storage disease, Gaucher disease, thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension)). In some embodiments, the methods described herein include: The present invention relates to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject having PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) or a disease or condition associated with PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).

[0185] In some embodiments, a polypeptide comprising an extracellular ActRIIA variant described herein reduces or inhibits the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors (e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA)). The polypeptides described herein may reduce the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors compared to the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors in the absence of the polypeptide of the present invention. In some embodiments, affecting myostatin, activin, and / or BMP9 signaling (e.g., decreasing or inhibiting the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors, e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA)) induces or increases red blood cell levels (e.g., hemoglobin levels, hematocrit levels, or red blood cell counts, e.g., red blood cell formation and / or red blood cell mass) in a subject. ), reducing fibrosis or the risk of developing fibrosis in a subject, delaying the onset of fibrosis, slowing or halting the progression of fibrosis, reversing fibrosis, reducing symptoms of PH in a subject (e.g., reduction in shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or heart palpitations, bluish color to the lips or skin (cyanosis), dizziness, or fainting), reducing the risk of developing PH in a subject, delaying the onset of PH, and / or slowing or halting the progression of PH. PH can be PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.

[0186] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), e.g., an effective amount of an ActRIIA variant) increases red blood cell levels (e.g., increases hemoglobin levels, increases hematocrit, increases red blood cell count, increases red blood cell mass, or induces or increases red blood cell formation), prevents or reduces fibrosis in a subject. The extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72), e.g., an effective amount of an ActRIIA variant) can increase red blood cell levels, prevent or reduce fibrosis, or prevent or treat PH (e.g., reduce fibrosis, prevent, delay, or attenuate the onset of fibrosis, slow or halt the progression of fibrosis, or reverse fibrosis), prevent or treat PH (e.g., reduce the symptoms of PH, prevent, delay, or attenuate the onset of PH, or slow or halt the progression of PH such as PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), or affect myostatin, activin, and / or BMP9 signaling. An extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72), e.g., an effective amount of an ActRIIA variant) can increase red blood cell levels, prevent or reduce fibrosis, or prevent or treat PH, compared to measurements obtained before treatment or compared to measurements obtained from an untreated subject with the same disease or condition. In some embodiments, the methods described herein do not cause vascular complications in the subject, such as increased vascular permeability or leakage. In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition involving low red blood cell levels (e.g., anemia or blood loss, e.g., anemia associated with cancer (e.g., multiple myeloma, leukemia, breast cancer, lung cancer, colon cancer), cancer treatment (e.g., chemotherapy or radiation therapy), myelodysplastic syndrome, chronic or acute kidney disease or failure (e.g., chronic kidney disease), inflammatory or autoimmune disease (e.g., rheumatoid arthritis, inflammatory bowel disease, e.g., Crohn's disease or ulcerative colitis, SLE), or surgery).In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition comprising fibrosis (e.g., chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis (e.g., cirrhosis), renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, osteoarthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumors, surgical adhesions, hypertrophic scars, keloids, or fibrosis associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease, heart disease, macular degeneration, retinal or vitreoretinopathy, systemic or localized scleroderma, atherosclerosis, or restenosis). In some embodiments of the methods described herein, the subject is diagnosed with PH (e.g., PAH, venous PH, hypoxemic PH, thromboembolic PH, or other PH, e.g., idiopathic PAH; hereditary PAH; PAH associated with or resulting from HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart abnormalities, connective tissue / autoimmune diseases (e.g., scleroderma and lupus), drug use or abuse (e.g., methamphetamine or cocaine use); venous PH associated with or resulting from left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), Hypoxic PH associated with or resulting from impaired alveolar ventilation, chronic exposure to high altitude, or developmental abnormalities; thromboembolic PH associated with or resulting from chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection); other PH associated with or resulting from blood disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic diseases (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (e.g., glycogen storage disease, Gaucher disease, thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).

[0187] The present invention also includes methods for treating a subject having or at risk of developing anemia or blood loss by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))). In any of the methods described herein, the subject has or is at risk of developing low red blood cell levels (e.g., low hemoglobin levels, or low red blood cell count, e.g., low red blood cell mass), and the subject has or is at risk of developing anemia or blood loss. In some embodiments, the anemia is caused by a nutritional deficiency (e.g., vitamin deficiency), bone marrow deficiency (e.g., paroxysmal nocturnal hemoglobinuria), an adverse reaction to a drug (e.g., antiretroviral HIV drugs), myelodysplastic syndrome, bone marrow transplant, cancer (e.g., a solid tumor such as breast cancer, lung cancer, or colon cancer; a lymphoid tumor such as chronic lymphocytic leukemia, non-Hodgkin's lymphoma, or Hodgkin's lymphoma; or a tumor of the hematopoietic system such as leukemia or multiple myeloma), cancer treatment (e.g., radiation or chemotherapy, e.g., chemotherapy with platinum-containing agents), an inflammatory or autoimmune disease (e.g., rheumatoid arthritis, other inflammatory arthritis, systemic lupus erythematosus (SLE), acute or chronic skin disease (e.g., psoriasis), or inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), cystitis, gastritis), acute or chronic kidney disease, including idiopathic or congenital conditions. Associated with or caused by disease or failure (e.g., chronic kidney disease), acute or chronic liver disease, diabetes, acute or chronic bleeding, infection (e.g., malaria, osteomyelitis), splenomegaly, porphyria, vasculitis, hemolysis, urinary tract infection, hemoglobinopathies (e.g., sickle cell disease), thalassemia, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Schwachman syndrome (e.g., Shwachman-Diamond syndrome), drug use or abuse (e.g., alcohol abuse), or transfusion contraindications (e.g., elderly patients, patients with alloantibodies or autoantibodies, pediatric patients, patients with cardiopulmonary disease, patients who object to transfusions for religious reasons (e.g., some Jehovah's Witnesses)).In some embodiments, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts. The compositions and methods described herein can also be used to treat subjects who do not adequately respond to erythropoietin (EPO) or who are susceptible to EPO's adverse effects (e.g., hypertension, headache, vascular thrombosis, flu-like syndrome, shunt occlusion, myocardial infarction). In some embodiments, the blood loss is due to surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding (e.g., menorrhagia). In some embodiments, the methods described herein increase red blood cell levels (e.g., hemoglobin level, hematocrit, or red blood cell count, e.g., red blood cell mass) compared to measurements taken before treatment. In some embodiments, the methods described herein increase or induce red blood cell formation compared to measurements taken before treatment. In some embodiments, the compositions and methods described herein reduce a subject's need for transfusions (e.g., the subject no longer requires transfusions, or the subject requires less frequent transfusions than before treatment with the compositions and methods described herein). Subjects with normal red blood cell levels can be treated using the methods and compositions described herein to increase red blood cell levels, thereby allowing blood to be collected and stored for later use in transfusions.

[0188] The present invention also includes methods for treating a subject having or at risk of developing fibrosis by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))). In any of the methods described herein, the subject has fibrosis or is at risk of developing fibrosis. In some embodiments, the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis (e.g., cystic fibrosis, idiopathic fibrosis, or fibrosis associated with tuberculosis, pneumonia, or coal dust), hepatic fibrosis (such as cirrhosis, biliary atresia, etc.), renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis (e.g., fibrosis associated with endomyocardial fibrosis or myocardial infarction), myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthrofibrosis, arthrofibrosis, tissue fibrosis (e.g., fibrosis affecting muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testes, ovaries, adrenal glands, arteries, veins, colon, small intestine, large intestine, biliary tract, or intestine), tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid. In some embodiments, the fibrosis is fibrosis associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments, the subject is at risk for developing fibrosis associated with cancer treatment (chemotherapy or radiation therapy), disease or infection (e.g., tuberculosis, pneumonia, myocardial infarction, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, restenosis), surgery, a wound, or a burn. In some embodiments, the methods described herein reduce fibrosis compared to measurements obtained before treatment or compared to fibrosis in an untreated subject.In some embodiments, the methods described herein prevent the onset of fibrosis, reduce the risk of developing fibrosis (e.g., reduce the risk of developing fibrosis compared to the onset of fibrosis in an untreated subject), or reverse existing fibrosis. In some embodiments, the methods described herein slow, halt, or reverse the progression of fibrosis (e.g., slow the progression of fibrosis compared to the progression before treatment compared to the progression in the absence of treatment or compared to the progression in an untreated subject). In some embodiments, the methods described herein improve organ or tissue function (e.g., the function of an organ or tissue with fibrosis) compared to the organ or tissue function before treatment. Tissue and organ function can be assessed using standard clinical tests commonly used to assess tissue and organ function.

[0189] The present invention also includes methods of treating a subject having or at risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72))). In any of the methods described herein, the subject has or is at risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, the PH is PAH. In some embodiments, the PAH is idiopathic PAH. In some embodiments, the PAH is hereditary PAH. In some embodiments, the PAH is PAH related to (e.g., caused by or associated with) HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, liver cirrhosis, congenital heart abnormalities, connective tissue / autoimmune diseases (e.g., scleroderma and lupus), or drug use or abuse (e.g., methamphetamine or cocaine use). In some embodiments, the PH is venous PH. In some embodiments, the venous PH is related to (e.g., caused by or associated with) left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathies, or congenital / acquired pulmonary vein stenosis. In some embodiments, the PH is hypoxic PH. In some embodiments, the hypoxic PH is hypoxic PH associated with (e.g., caused by or related to) chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorder, chronic exposure to high altitude, or developmental abnormalities. In some embodiments, the PH is thromboembolic PH.In some embodiments, the thromboembolic PH is thromboembolic PH related to (e.g., caused by or associated with) chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection). In some embodiments, the PH is other PH. In some embodiments, the other PH is other PH related to (e.g., caused by or associated with) a blood disease (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension. In some embodiments, the methods described herein reduce symptoms of PH compared to the frequency or severity of the symptoms before treatment (e.g., reduce the frequency or severity of symptoms such as shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness, or fainting). In some embodiments, the methods described herein prevent the onset of PH or reduce the risk of developing PH (e.g., reduce the risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) compared to the onset of PH in an untreated subject). In some embodiments, the methods described herein slow or halt the progression of PH (e.g., slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) compared to the progression before treatment or compared to the progression in an untreated or untreated subject). In some embodiments, the methods described herein reduce pulmonary vascular remodeling or vascular remodeling in the heart (e.g., the onset or progression of vascular remodeling in the heart or lungs) in a subject compared to vascular remodeling before treatment or compared to vascular remodeling in an untreated subject.In some embodiments, the methods described herein reduce right ventricular hypertrophy (e.g., reduce right ventricular hypertrophy or reduce the progression of right ventricular hypertrophy) compared to the right ventricle before treatment or compared to right ventricular hypertrophy in an untreated subject. Symptoms of PH can be assessed before and after treatment using standard clinical tests. Tests commonly used to assess PH include electrocardiograms, pulmonary function tests, echocardiograms, right heart catheterization, computed tomography scans, measurement of pulmonary vascular resistance, and 6-minute walk tests. In some embodiments, the methods described herein reduce pulmonary vascular resistance (e.g., result in a reduction in pulmonary vascular resistance compared to pre-treatment pulmonary vascular resistance). In some embodiments, the methods described herein improve performance in a 6-minute walk test compared to performance in a 6-minute walk test before treatment.

[0190] In any of the methods described herein, a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs: 6-71)) and further comprising a C-terminal extension of 1 to 6 amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids) can be used as a therapeutic protein. In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) formed by the interaction of two Fc domain monomers, each fused to a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be used 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 the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be used as a therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing the nucleic acids, may also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector can be administered as part of a pharmaceutical composition. Compositions that can be administered to a subject according to the methods described herein are provided in Table 4 below.

[0191] [Table 4-1]

[0192] [Table 4-2]

[0193] [Table 4-3]

[0194] [Table 4-4]

[0195] [Table 4-5]

[0196] [Table 4-6]

[0197] [Table 4-7] [Example]

[0198] Example 1: Evaluation of ActRIIA variant binding affinity by surface plasmon resonance (SPR) Biacore3000 was used to measure the kinetics of interactions between ActRIIA variants and their ligands, activin A, activin B, growth differentiation factor 11 (GDF11), and BMP-9. ActRIIA variants were generated by transient expression in HEK293 cells and purified from conditioned medium using protein A-Sepharose chromatography. ActRIIA variants were immobilized on chips (CM4 or CM5) using a capture antibody (anti-mouse from GE Healthcare) in flow cells 2–4 to ensure proper orientation. Flow cell 1 was used as a reference cell to subtract nonspecific binding and bulk effects. HBS-EP+ buffer (GE Healthcare™) was used as the running buffer. Each ligand was flowed at a constant concentration series at 40 μl / min to avoid mass transfer effects. The K for each interaction was calculated. DData were analyzed using Scrubber2 with BioLogic™ software to calculate (Table 5).

[0199] [Table 5]

[0200] Example 2 - Effect of ActRIIA / B-Fc on fibrosis in mdx mice We analyzed the effect of ActRIIA / B-Fc (SEQ ID NO: 69 fused to the Fc domain) on reducing intramuscular fibrosis associated with muscular dystrophy using mdx mice, a mouse model of Duchenne muscular dystrophy. Briefly, 12- to 13-month-old female C57Bl / 10 and mdx mice were treated twice weekly for 12 weeks with vehicle or 20 mg / kg ActRIIA / B-Fc. Diaphragms and quadriceps were harvested at necropsy and immediately flash-frozen in liquid nitrogen. Protein was extracted from the tissues and hydrolyzed prior to high-performance liquid chromatography on a Hitachi L 8900 amino acid analyzer. Individual amino acid content was determined by comparing chromatographic peaks with known standards. Hydroxyproline, a collagen-specific amino acid and surrogate for total fibrosis content, was calculated as mg per gram of total protein extracted. As shown in Figure 2, treatment with ActRIIA / B-Fc attenuated the development of fibrosis in aged mdx mice.

[0201] Example 3 - Effect of ActRIIA / B-Fc on red blood cell mass in non-human primates Two- to three-year-old cynomolgus monkeys were administered vehicle or 3, 10, or 50 mg / kg of ActRIIA / B-Fc (SEQ ID NO: 69 fused to the Fc domain) every other week for three months. On day 92, blood was collected in K2EDTA tubes and analyzed on an Advia hematology analyzer. As shown in Figure 3, treatment with ActRIIA / B-Fc dose-dependently increased red blood cell mass in non-human primates. Hct - hematocrit, Hgb - hemoglobin, RBC - red blood cell count.

[0202] Example 4: Effect of extracellular ActRIIA mutants on PAH In one experiment, male rats were induced with a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg) to induce PAH. To determine whether treatment with ActRIIA mutants could prevent the development of PAH, rats were randomized to vehicle or ActRIIA mutant treatment groups 24 h after PAH induction and treated twice weekly with ActRIIA mutants (5 or 15 mg / kg) or vehicle for 21 days. On day 14, rats were anesthetized with 1.5% isoflurane while held in a supine position. Electrocardiograms were used to examine pulmonary blood flow acceleration, right ventricular function and hypertrophy, and left ventricular function, and right ventricular (RV) remodeling was examined using a small-animal high-frequency ultrasound probe. Doppler imaging across the mitral and tricuspid valves was used to determine whether treatment with ActRIIA mutants induced obvious regurgitation or lesions. On day 21, rats were anesthetized with pentobarbital, intubated via the trachea, and mechanically ventilated using a rodent ventilator. Hemodynamics was assessed using a fluid-filled catheter inserted through the right ventricular apex. Rats were perfused with PBS followed by 1% paraformaldehyde (PFA). To measure right ventricular hypertrophy (RVH), hearts were removed, and the right ventricular free wall was dissected from the left ventricle plus septum (LV+S) and weighed separately. The degree of RVH was determined from the ratio RV / (LV+S).

[0203] In the second experiment, male rats were given a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg) to induce PAH. To determine whether treatment with ActRIIA mutants could slow or reduce the progression of PAH, rats were reinjected with MCT on day 18 and randomized to vehicle or ActRIIA mutant treatment groups. Rats were injected with ActRIIA mutants (15 mg / kg) or vehicle three times weekly. Hemodynamics and RVH were examined on day 35 as described above.

[0204] Example 5: Treatment of anemia by administration of extracellular ActRIIA mutants According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, with anemia (e.g., vitamin deficiency anemia or anemia associated with chronic kidney disease) to increase parameters of red blood cell mass, such as red blood cell count, hemoglobin level, or hematocrit. Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on a blood test measuring a hematological parameter. To treat a subject, a physician skilled in the art can administer a composition containing an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) to the subject. The composition containing the extracellular ActRIIA variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat anemia. The extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIA variant is administered in an amount sufficient to increase hemoglobin levels, increase red blood cell count, or increase hematocrit.

[0205] After administering the composition to a patient, those skilled in the art can monitor the patient's improvement in response to treatment by various methods.For example, a doctor can monitor the patient's hemoglobin level, red blood cell count, or hematocrit by performing blood tests.Compared to the test results before administering the composition, the discovery that the patient's hemoglobin level, red blood cell count, or hematocrit improves after administering the composition indicates that the patient is responding favorably to treatment.Subsequent doses can be determined and administered as needed.

[0206] Example 6 - Treatment of fibrosis by administration of extracellular ActRIIA mutants According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, with fibrosis (e.g., pulmonary fibrosis or fibrosis associated with chronic kidney disease) to reduce the symptoms of fibrosis or slow or halt the progression of fibrosis. Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on a clinical test for fibrosis (e.g., an imaging test such as an X-ray or CT scan). To treat a subject, a physician skilled in the art can administer a composition containing an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) to the subject. The composition containing the extracellular ActRIIA variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat fibrosis, or can be administered locally (e.g., injected) into a fibrotic tissue or organ. The extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIA variant is administered in an amount sufficient to reduce the symptoms of fibrosis or to slow or stop the progression of fibrosis.

[0207] After administering the composition to a patient, those skilled in the art can monitor the improvement of the patient in response to treatment by various methods.For example, a doctor can perform imaging tests to monitor the patient's fibrosis and use standard clinical tests to monitor the patient's symptoms.Compared to the test results before administering the composition, the discovery that the patient's symptoms are reduced or the progression of the patient's fibrosis is delayed or stopped after administering the composition indicates that the patient is responding favorably to treatment.Subsequent doses can be determined and administered as needed.

[0208] Example 7 - Treatment of pulmonary hypertension by administration of extracellular ActRIIA variants According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, with pulmonary hypertension (PH, e.g., PAH) to reduce the symptoms of PH or slow or stop the progression of PH. The treatment method can include diagnosing or identifying the subject as a candidate for treatment based on standard clinical tests for PH (e.g., echocardiogram, electrocardiogram, chest X-ray, right heart catheterization). To treat the subject, a physician skilled in the art can administer to the subject a composition containing an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)). The composition containing the extracellular ActRIIA variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat PH. The extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIA variant is administered in an amount sufficient to reduce the symptoms of PH or to slow or stop the progression of PH.

[0209] After administering the composition to the patient, those skilled in the art can monitor the patient's improvement in response to treatment using various methods. For example, a physician can monitor the patient's condition using standard clinical tests and patient self-reporting. A finding that the patient's condition after administration of the composition has reduced PH symptoms or the progression of the patient's PH has slowed or stopped compared to the test results before administration of the composition indicates that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0210] Other embodiments While the invention has been described in terms of particular embodiments thereof, it will be understood that further modifications are possible, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow from the principles of the invention, including such departures from the present disclosure as are within known or related practice within the art to which this invention pertains and which may fall within the essential characteristics set forth above.

[0211] The entire contents of all publications, patents, and patent applications are incorporated herein by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0212] Other embodiments are within the scope of the following claims. The technical concepts that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] A method of treating a subject having or at risk of developing fibrosis, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0213] [Appendix 2] A method of reducing or preventing fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0214] [Appendix 3] A method of slowing, inhibiting, or reversing fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0215] [Appendix 4] A method for attenuating the development of fibrosis, comprising administering to a subject a therapeutically effective amount of a polypeptide of a composition of Table 4. [Appendix 5] A method for ameliorating fibrosis, comprising administering to a subject a therapeutically effective amount of a polypeptide of a composition of Table 4.

[0216] [Appendix 6] A method for affecting at least one of myostatin, activin, and BMP9 signaling in a subject having or at risk of developing fibrosis, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0217] [Appendix 7] The method according to any one of Appendices 1 to 6, wherein the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, corneal fibrosis, cardiac fibrosis, bone marrow fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthrofibrosis, arthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid.

[0218] [Appendix 8] The method described in Appendix 7, wherein the tissue fibrosis is fibrosis affecting a tissue selected from the group consisting of muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine, large intestine, biliary tract, and intestine.

[0219] [Appendix 9] The method of any one of Appendices 1 to 6, wherein the fibrosis is associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease, chronic kidney disease, heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis.

[0220] [Appendix 10] The method according to any one of Appendices 1 to 9, wherein the method improves the function of a fibrotic tissue or organ. [Appendix 11] A method of increasing red blood cell levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0221] [Appendix 12] A method of promoting or increasing red blood cell formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0222] [Appendix 13] The method of Appendices 11 and 12, wherein the subject has or is at risk of developing anemia or blood loss. [Appendix 14] A method of affecting at least one of myostatin, activin, and BMP9 signaling in a subject having or at risk of developing a disease or condition involving low red blood cell levels, low hematocrit, or low hemoglobin levels, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0223] [Appendix 15] The method of Appendices 14, wherein the disease or condition is anemia or blood loss. [Appendix 16] The method of Appendices 13 and 15, wherein the anemia or blood loss is associated with cancer, cancer treatment, chronic kidney disease, acute kidney disease or renal failure, chronic kidney disease or renal failure, myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse drug reaction, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, acute liver disease, chronic liver disease, diabetes, acute bleeding, chronic bleeding, infection, hemoglobinopathies, drug use, alcohol abuse, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Schwachman syndrome, advanced age, contraindications to blood transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0224] [Appendix 17] The method of any one of Appendices 13, 15, or 16, wherein the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0225] [Appendix 18] A method of treating a subject having or at risk of developing anemia, comprising administering to the subject a therapeutically effective amount of a composition of Table 4. [Appendix 19] The method of Appendices 18, wherein the anemia is associated with cancer, cancer treatment, chronic kidney disease, acute kidney disease or renal failure, chronic kidney disease or renal failure, myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse drug reaction, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, acute liver disease, chronic liver disease, diabetes, acute bleeding, chronic bleeding, infection, hemoglobinopathies, drug use, alcohol abuse, advanced age, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Schwachman syndrome, contraindications to blood transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0226] [Appendix 20] The method of Appendices 18 and 19, wherein the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0227] [Appendix 21] The method of any one of Appendices 11 to 20, wherein the subject does not respond well to treatment with erythropoietin (EPO) or is sensitive to the side effects of EPO.

[0228] [Appendix 22] The method of any one of Appendices 11 to 21, wherein the method increases erythropoiesis, red blood cell count, hematocrit, or hemoglobin levels. [Appendix 23] The method of any one of Appendices 11 to 22, wherein the method reduces the subject's need for blood transfusions.

[0229] [Appendix 24] A method for preventing pulmonary hypertension (PH) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0230] [Appendix 25] A method for slowing or inhibiting the progression of PH in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0231] [Appendix 26] A method of treating a subject having or at risk of developing PH, comprising administering to the subject a therapeutically effective amount of a composition of Table 4. [Appendix 27] A method of affecting at least one of myostatin, activin, and BMP9 signaling in a subject having or at risk of developing PH, comprising administering to the subject a therapeutically effective amount of a composition of Table 4.

[0232] [Appendix 28] The method according to any one of Appendices 24 to 27, wherein the PH is pulmonary arterial hypertension (PAH). [Appendix 29] The method described in Appendix 28, wherein the PAH is idiopathic PAH.

[0233] [Appendix 30] The method described in Appendix 28, wherein the PAH is hereditary PAH. [Appendix 31] The method of Appendices 28, wherein the PAH is associated with HIV infection, schistosomiasis, cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, autoimmune disorders, or drug use or abuse.

[0234] [Appendix 32] The method according to any one of Appendices 24 to 27, wherein the PH is venous PH. [Appendix 33] The method of Appendices 32, wherein the venous PH is associated with left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital or acquired pulmonary vein stenosis.

[0235] [Appendix 34] The method according to any one of Appendices 24 to 27, wherein the PH is hypoxic PH. [Appendix 35] The method of Appendices 34, wherein the hypoxic PH is associated with chronic obstructive pulmonary disease, interstitial lung disease, sleep-disordered breathing, pulmonary fibrosis, alveolar hypoventilation disorder, chronic exposure to high altitude, or a developmental abnormality.

[0236] [Appendix 36] The method according to any one of Appendices 24 to 27, wherein the PH is thromboembolic PH. [Appendix 37] The method of Appendices 36, wherein the thromboembolic PH is associated with chronic thromboembolic pulmonary hypertension, pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection.

[0237] [Appendix 38] The method according to any one of Appendices 24 to 27, wherein the PH is another PH. [Appendix 39] The method of Appendices 38, wherein the other PH is associated with a blood disorder, a systemic disease, a metabolic disorder, pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0238] [Appendix 40] The method of any one of Appendices 24 to 39, wherein the method reduces the frequency or severity of one or more symptoms of PH. [Appendix 41] The method of any one of Appendices 24 to 40, wherein the method reduces pulmonary vascular remodeling or vascular remodeling in the subject's heart.

[0239] [Appendix 42] The method of any one of Appendices 24 to 41, wherein the method reduces right ventricular hypertrophy. [Appendix 43] The method of any one of Appendices 24 to 42, wherein the method reduces pulmonary vascular resistance.

[0240] [Appendix 44] The method described in any one of Appendices 24 to 43, wherein the method improves performance in a 6-minute walk test. [Appendix 45] The method of any one of Appendices 1 to 44, wherein the method reduces or inhibits binding of at least one of activin and myostatin to their endogenous receptors.

[0241] [Appendix 46] The method of any one of Appendices 1-10 and 45, wherein the composition is administered in an amount sufficient to reduce fibrosis, prevent fibrosis, reduce the risk of developing fibrosis, delay or attenuate the onset of fibrosis, delay, inhibit or reverse the progression of fibrosis, reverse fibrosis, treat fibrosis, reduce one or more symptoms of fibrosis, improve the function of a fibrotic tissue or organ, affect the signaling of at least one of myostatin, activin, and BMP9 in a subject, or reduce or inhibit the binding of at least one of activin and myostatin to their endogenous receptors.

[0242] [Appendix 47] The method of any one of Appendices 11-23 and 45, wherein the composition is administered in an amount sufficient to increase red blood cell levels, increase hemoglobin levels, increase red blood cell formation, increase red blood cell count, increase hematocrit, reduce the need for blood transfusions, treat anemia, affect signaling of at least one of myostatin, activin, and BMP9 in the subject, or reduce or inhibit binding of at least one of activin and myostatin to their endogenous receptors.

[0243] [Appendix 48] The method of any one of Appendices 24-45, wherein the composition is administered in an amount sufficient to prevent PH, reduce the risk of developing PH, reduce the severity or frequency of one or more symptoms of PH, delay the onset of PH, delay or inhibit the progression of PH, treat PH, reduce pulmonary vascular remodeling, reduce cardiac vascular remodeling, reduce right ventricular hypertrophy, reduce pulmonary vascular resistance, improve performance in a 6-minute walk test, affect signaling of at least one of myostatin, activin, and BMP9 in a subject, or reduce or inhibit binding of at least one of activin and myostatin to their endogenous receptors.

[0244] [Appendix 49] The method of any one of Appendices 1 to 48, wherein the method does not cause vascular complications in the subject. [Appendix 50] The method of Appendices 49, wherein the method does not increase vascular permeability or leakage.

Claims

1. A pharmaceutical composition for treating a subject having or at risk of developing anemia associated with myelodysplastic syndrome or anemia associated with myelofibrosis, the pharmaceutical composition comprising a therapeutically effective amount of a polypeptide, the polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant having the sequence of SEQ ID NO: 69 and fused at its C-terminus to a human IgG1 Fc domain monomer, the polypeptide being in the form of a dimer.

2. The pharmaceutical composition of claim 1 , wherein the human IgG1 Fc domain monomer is fused to the C-terminus of the variant via a linker.

3. The linker may be selected from the group consisting of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 98), GGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 100), GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), SGGG (SEQ ID NO: 107), GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGGA (SEQ ID NO: 112), GSGSGSGSG (SEQ ID NO: 113), GAGAGAGAG (SEQ ID NO: 114), GSGSGSGSG (SEQ ID NO: 115), GAGAGAGAGAG (SEQ ID NO: 116), GSGSGSGSGSG (SEQ ID NO: 117), GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), GGSGGSGGSGGGS (SEQ ID NO: 123), GGAGGGAG (SEQ ID NO: 124), GGSGGGGSG (SEQ ID NO: 125) 5), GGAGGGAGGGAG (SEQ ID NO: 126), GGSGGGSGGGSG (SEQ ID NO: 127), GGGGAGGGGAGGGGA (SEQ ID NO: 128), GGGGSGGGGSGGGGS (SEQ ID NO: 129), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), GGGAGGG (SEQ ID NO: 132), AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), No. 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), GGSGGGSEGGGGSEGGGGSEGGGGSEGGGGSEGGGGSGGGS (SEQ ID NO: 146), EAAAK (SEQ ID NO: 147),or PAPAP (SEQ ID NO: 148).

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