Activin receptor type IIA variants and methods of use thereof

Abstract

The present invention features polypeptides comprising extracellular ActRIIA variants. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIA variant fused to an Fc domain monomer or portion. The present invention also features pharmaceutical compositions and methods for using the polypeptides to treat diseases or conditions involving low red blood cell levels (e.g., anemia or blood loss; fibrosis; or pulmonary hypertension).

Description

Background of the Invention

[0002] Fibrosis is the formation of excess connective tissue in an organ or tissue. The connective tissue that can be formed in response to damage (such as damage) or as part of an immune response (such as inflammatory response) can destroy the structure and function of the organ or tissue in which it is formed, resulting in an increase in tissue hardness. Fibrosis can occur in many organs and tissues in vivo, especially including lung (such as pulmonary fibrosis, cystic fibrosis), liver (such as cirrhosis), heart (such as endomyocardial fibrosis or post-myocardial infarction fibrosis), brain (such as glial cell scar formation), skin (such as keloid formation), kidney (such as renal fibrosis) and eye (such as corneal fibrosis); and it is known to be relevant to certain drug treatments (such as chemotherapy, radiotherapy and surgery). There are limited treatment options for patients with fibrosis, and most treatments focus on improving quality of life or temporarily slowing down disease progression.

[0003] Anemia is a global health problem, with health implications that affect both morbidity and mortality. Only in the U.S., the prevalence of anemia almost doubled from 2003 to 2012. The symptoms of anemia include fatigue, weakness, shortness of breath, palpitations and cognitive decline, and it has been found that children, pregnant women, women of childbearing age and the elderly have the highest risk of developing anemia. The most common form of anemia is iron deficiency anemia, but anemia can also be caused by chronic diseases, blood loss and red blood cell destruction. Although 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 need blood transfusion.

[0004] Pulmonary hypertension (PH) is a serious condition characterized by higher-than-normal pressure in the blood vessels between the lungs and the heart. PH can be classified into five main types: arterial (PAH), venous (PH secondary to left-sided heart disease), hypoxia (PH caused by lung disease), thromboembolic (PH caused by chronic arterial obstruction, such as blood clots), or other (PH with unclear or multifactorial mechanisms), also known as WHO Class IV. PAH is characterized by increased pulmonary vascular pressure caused by scarring, which occurs when small blood vessels in the lungs become blocked or narrowed. This leads to increased resistance to blood flow through the lungs and forces the right side of the heart to work harder, which can lead to heart failure, reduced blood oxygenation, and shortened life expectancy. PAH can be idiopathic (e.g., with no identifiable cause), heritable (e.g., familial, often due to genetic mutations), or may be associated with drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital heart anomalies, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus). Treatments for PH include vasodilators, anticoagulants, and supplemental oxygen, but these therapies manage disease symptoms rather than targeting the biological mechanisms that cause the disease.

[0005] There is a need for new treatments for fibrosis, anemia, and PH. SUMMARY OF THE INVENTION

[0007] The present invention features polypeptides comprising extracellular activin receptor type IIA (ActRIIA) variants. In some embodiments, the polypeptides of the present invention comprise extracellular ActRIIA variants fused to the N- or C-terminus of an Fc domain monomer or portion. Such portions can be linked by amino acids or other covalent bonds and can increase the stability of the polypeptide. Polypeptides comprising extracellular ActRIIA variants fused to Fc domain monomers can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The polypeptides of the present invention can also 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. The polypeptides of the present invention can also be used 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) in a subject in need thereof, e.g., a subject having 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, 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. In addition, the polypeptides of the present invention can be used to treat, prevent, delay, or attenuate the development or progression of pulmonary hypertension in a subject having pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or other pulmonary hypertension) or at risk of developing pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or other pulmonary hypertension). Further, the polypeptides of the invention can also be used to influence myostatin, activin and / or bone morphogenetic protein 9 (BMP9) signaling in subjects who have or are 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).

[0008] In one aspect, the invention features a polypeptide comprising an extracellular activin receptor type IIA (ActRIIA) variant having the sequence:

[0009] GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX16 NISGSIEIV

[0010] X 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: 1), wherein X1 is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 It is K or Q; X 11 It is D or A; X 12 It is K or A; X 13 is R or A; X 14 Is R or L; X 15 Is F or Y; X 16 is K, R or A; X 17 is K, A, Y, F or I; X 18 It is Q or K; X 19 Is W or A; X 20 L or A; X 21 is D, K, R, A, F, G, M, N, or I; X 22 Is I, F or A; X 23 Is K or T; X 24 Is K or E; X 25 Is D or E; X 26 is S or N; and X 27 is E or Q, and wherein the variant has at least one amino acid substitution relative to wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73 or extracellular ActRIIA having the sequence of any one of SEQ ID NOs: 76-96.

[0011] In some embodiments, the variant has the following sequence:

[0012] GAILGRSETQECLFX2NANWX3X4X5X6TNQTGVEX7CX8GX9KX 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX17

[0013] 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), where X2, X3, X4, X5, X6, X7, X8, X9, 11 、X 12 、X 13 、X 14 、X 15 、X 16 、X 17 、X 18 、X 19 、X 20 、X 21 、X 22 、X 23 、X 24 、X 25 、X 26 and X 27 As defined above.

[0014] In some embodiments, the variant has the following sequence:

[0015] GAILGRSETQECLFX2NANWEX4X5RTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3), wherein X2, X4, X5, X7, X8, X9, X 14 、X 15 、X 16 、X 18 、X 22 、X 23 、X 24 、X 25 、X26 and X 27 As defined above.

[0016] In some embodiments, the variant has the following sequence:

[0017] 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 As defined above.

[0018] In some embodiments, the variant has the following sequence:

[0019] GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14 HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5), wherein X2, X4, X8, X9, X 14 、X 18 、X 23 、X 25 、X 26 and X 27 As defined above.

[0020] In any of the preceding embodiments, X1 is F or Y. In any of the preceding embodiments, X2 is F or Y. In any of the preceding embodiments, X3 is E or A. In any of the preceding embodiments, X4 is K or L. In any of the preceding embodiments, X5 is D or E. In any of the preceding embodiments, X6 is R or A. In any of the preceding embodiments, X7 is P or R. In any of the preceding embodiments, X8 is Y or E. In any of the preceding embodiments, X9 is D or E. In any of the preceding embodiments, X 10 is K or Q. In any of the foregoing embodiments, X 11 is D or A. In any of the foregoing embodiments, X 12 is K or A. In any of the foregoing embodiments, X 13 is R or A. In any of the foregoing embodiments, X 14 is R or L. In any of the foregoing embodiments, X 15 is F or Y. In any of the foregoing embodiments, X 16 is K, R or A. In any of the foregoing embodiments, X 17 is K, A, Y, F or I. In any of the foregoing embodiments, X 18 is Q or K. In any of the foregoing embodiments, X 19 is W or A. In any of the foregoing embodiments, X 20 is L or A. In any of the foregoing embodiments, X 21 is D, K, R, A, F, G, M, N, or I. In any of the foregoing embodiments, X 22 is I, F or A. In any of the foregoing embodiments, X 23 is K or T. In any of the foregoing embodiments, X 24 is K or E. In any of the foregoing embodiments, X 25 is D or E. In any of the foregoing embodiments, X 26 is S or N. In any of the foregoing embodiments, X 27 is E or Q. In any of the foregoing embodiments, X 23 It's T, X 24 It's E, X 25 is E, and X 26 is N. In any of the foregoing embodiments, X 23 It's T, X 24 It's K, X 25 is E, and X 26 is N. In any of the foregoing embodiments, X 17 It’s K.

[0021] In any of the preceding embodiments, the variant has the sequence of any one of SEQ ID NOs: 6-72.

[0022] In any of the preceding embodiments, position X 24 The amino acid at the position can be replaced by amino acid K.

[0023] In any of the preceding embodiments, position X 24 The amino acid at the position can be replaced by amino acid E.

[0024] In any of the foregoing embodiments, the polypeptides described herein can further comprise a C-terminal extension of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids). In some embodiments, the C-terminal extension is the amino acid sequence NP. In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 155).

[0025] In any of the foregoing embodiments, the polypeptides described herein may further comprise a moiety fused or covalently linked to the C-terminus of the polypeptide. In some embodiments, the moiety increases the stability of the polypeptide or improves the pharmacokinetics of the polypeptide. In some embodiments, the moiety is an Fc domain, an albumin binding peptide, a fibronectin domain, or human serum albumin.

[0026] In any of the foregoing embodiments, the polypeptide described herein may further comprise an Fc domain monomer fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the polypeptide comprising an extracellular ActRIIA variant described herein fused to an Fc domain monomer can 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.

[0027] In any of the foregoing embodiments, the polypeptide described herein may further comprise an Fc domain fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the Fc domain is a wild-type Fc domain. In some embodiments, the wild-type Fc domain has the sequence of SEQ ID NO: 151. In some embodiments, the Fc domain contains one or more amino acid substitutions. In some embodiments, the Fc domain containing one or more amino acid substitutions does not form a dimer.

[0028] In any of the foregoing embodiments, the polypeptide described herein may further comprise an albumin binding peptide fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the albumin binding peptide has the sequence of SEQ ID NO: 152.

[0029] In any of the foregoing embodiments, the polypeptide described herein may further comprise a fibronectin domain fused to the C-terminus of the polypeptide by way of a linker.In some embodiments, the fibronectin domain peptide has the sequence of SEQ ID NO:153.

[0030] In any of the foregoing embodiments, the polypeptide described herein may further comprise human serum albumin fused to the C-terminus of the polypeptide by way of a linker. In some embodiments, the human serum albumin has a sequence of SEQ ID NO: 154.

[0031] In some embodiments, the linker is an amino acid spacer. In some embodiments, the amino acid spacer is GGG, GGGA (SEQ ID NO:98), GGGG (SEQ ID NO:100), GGGAG (SEQ ID NO:130), GGGAGG (SEQ ID NO:131), or GGGAGGG (SEQ ID NO:132).

[0032] In some embodiments, the amino acid spacer is GGGS (SEQ ID NO:99), GGGGA (SEQ ID NO:101), GGGGS (SEQ ID NO:102), GGGGG (SEQ ID NO:103), GGAG (SEQ ID NO:104), GGSG (SEQ ID NO:105), AGGG (SEQ ID NO:106), SGGG (SEQ ID NO:107), GAGA (SEQ ID NO:108), GSGS (SEQ ID NO:109), GAGAGA (SEQ ID NO:110), GSGSGS (SEQ ID NO:111), GAGAGAGA (SEQ ID NO:112), GSGSGSGS (SEQ ID NO:113), GAGAGAGAGA (SEQ ID NO:114), GSGSGSGSGS (SEQ ID NO:115), GAGAGAGAGAGA (SEQ ID NO:116), and GSGSGSGSGSGS (SEQ ID NO:117), GGAGGA (SEQ ID NO:118), GGSGGS (SEQ ID NO:119), GGAGGAGGA (SEQ ID NO:120), GGSGGSGGS (SEQ ID NO:121), GGAGGAGGAGGA (SEQ ID NO:122), GGSGGSGGSGGS (SEQ ID NO:123), GGAGGGAG (SEQ ID NO:124), GGSGGGSG (SEQ ID NO:125), GGAGGGAGGGAG (SEQ ID NO:126), and GGSGGGSGGGSG (SEQ ID NO:127), GGGGGAGGGGAGGGGA (SEQ ID NO:128), GGGGSGGGGSGGGGS (SEQ ID NO:129), AAAL (SEQ ID NO:133), AAAK (SEQ ID NO:134), AAAR (SEQ ID NO:135), EGKSSGSGSESKST (SEQ ID NO:136), GSAGSAAGSGEF (SEQ ID NO:137), AEAAAKEAAAKA (SEQ ID NO:138), KESGSVSSEQLAQFRSLD (SEQ ID NO:139), GENLYFQSGG (SEQ ID NO:140), SACYCELS (SEQ IDNO:141), RSIAT (SEQ ID NO:142), RPACKIPNDLKQKVMNH (SEQ ID NO:143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:144), AAANSSIDLISVPVDSR (SEQ ID NO:145), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:144) NO:146), EAAAK (SEQ ID NO:147) or PAPAP (SEQ ID NO:148).

[0033] In any of the foregoing embodiments, the polypeptide described herein has a serum half-life (eg, in a human subject) of at least 7 days.

[0034] In any of the preceding embodiments, the polypeptides described herein are expressed with a K of 200 pM or greater. D Binds to human bone morphogenetic protein 9 (BMP9). In some embodiments, the polypeptide binds to activin and / or myostatin and has reduced (e.g., weak) binding to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0035] In any of the preceding embodiments, the polypeptides described herein have a K of 800 pM or less. D Binds human activin A.

[0036] In any of the foregoing embodiments, the polypeptides described herein have a K of approximately 800 pM or less. D Binds human activin B.

[0037] In any of the foregoing embodiments, the polypeptides described herein have a K of approximately 5 pM or greater. D Binds human GDF-11.

[0038] In another aspect, the invention features a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72). In another aspect, the invention also features a vector comprising a nucleic acid molecule described herein.

[0039] In another aspect, the invention features a host cell that expresses a polypeptide described herein, wherein the host cell comprises a nucleic acid molecule or vector described in the first two aspects, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0040] In another aspect, the invention features a method of making a polypeptide described herein, wherein the method includes: a) providing a host cell comprising a nucleic acid molecule or vector described herein, and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow formation of the polypeptide.

[0041] In another aspect, the invention features 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.

[0042] In another aspect, the invention also features 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- or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0043] In another aspect, the invention also features a construct (e.g., a heterodimer) comprising two different 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- or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0044] In another aspect, the invention features 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.

[0045] In another aspect, the invention features 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.

[0046] In another aspect, the invention features 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.

[0047] In another aspect, the invention features 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.

[0048] In another aspect, the invention features a method of attenuating 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.

[0049] In another aspect, the invention features 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.

[0050] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing fibrosis, 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.

[0051] In some embodiments of any of the aforementioned aspects, the fibrosis is chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarticular fibrosis, arthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumors, surgical adhesions, hypertrophic scars, or keloids. 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, neural tissue, testicles, ovaries, adrenal glands, arteries, veins, colon, small intestine, large intestine, bile duct, and intestine.

[0052] In some embodiments of any of the preceding aspects, the fibrosis is associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis 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 preceding aspects, the fibrosis is caused by chronic kidney disease.

[0053] In some embodiments of any of the aforementioned aspects, the method improves the function of fibrotic tissue or organs. In some embodiments of any of the aforementioned aspects, the method slows, inhibits, or reverses the progression of fibrosis. In some embodiments of any of the aforementioned aspects, the method reduces (e.g., reduces the frequency or severity of) or reverses one or more symptoms of fibrosis.

[0054] In another aspect, the invention features 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.

[0055] In another aspect, the invention features 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.

[0056] In some embodiments of any of the aforementioned aspects, the subject has or is at risk of developing anemia or blood loss.

[0057] In another aspect, the invention features a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having a disease or condition, or at risk of developing a disease or condition, 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.

[0058] In another aspect, the invention features a method of treating a subject having anemia or at risk of developing anemia, 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.

[0059] In some embodiments of any of the preceding aspects, the anemia or blood loss is associated with cancer, cancer treatment, kidney disease or 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 defect, bone marrow transplantation, liver disease (e.g., acute liver disease or chronic liver disease), diabetes, bleeding (e.g., acute or chronic bleeding), infection, hemoglobinopathy, drug use, alcohol abuse, advanced age, Churg-Felty syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schonlein purpura, Schwakman syndrome (e.g., Schwakman-Dieter-Lysson syndrome), a contraindication to blood transfusion, surgery, trauma, a wound, an ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donation, or excessive menstrual bleeding.

[0060] In some embodiments of any of the preceding aspects, the anemia is caused by chronic kidney disease.

[0061] In some embodiments of any of the preceding 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, DeBouchet-Brown anemia, Fanconi anemia, or refractory anemia with blastosis.

[0062] In some embodiments of any of the aforementioned aspects, the subject does not respond well to treatment with erythropoietin (EPO), or is susceptible to adverse effects of EPO.

[0063] In some embodiments of any of the aforementioned aspects, the method increases red blood cell formation, red blood cell count, hematocrit, or hemoglobin levels (eg, red blood cell mass).

[0064] In some embodiments of any of the aforementioned aspects, the method reduces the subject's need for blood transfusion.

[0065] In another aspect, the invention features 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.

[0066] In another aspect, the invention features 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.

[0067] In another aspect, the invention features 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.

[0068] In another aspect, the invention features a method of treating a subject having PH 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.

[0069] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having PH 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.

[0070] In another aspect, the invention features a method of reducing vascular remodeling in a subject having PH 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.

[0071] In another aspect, the invention features a method of reducing right ventricular hypertrophy in a subject having PH 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.

[0072] In another aspect, the invention features a method of reducing pulmonary vascular resistance in a subject having PH 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.

[0073] In some embodiments of any of the aforementioned aspects, the PH is pulmonary arterial hypertension (PAH). In some embodiments, the PH is idiopathic PAH. In some embodiments, the PH is heritable PAH. In some embodiments, the PAH is associated with HIV infection, schistosomiasis, cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary angiomatosis, connective tissue disorders, autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., cocaine or methamphetamine use).

[0074] In some embodiments of any of the aforementioned aspects, the PH is venous PH. In some embodiments, 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.

[0075] In some embodiments of any of the aforementioned 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), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitude, or developmental abnormalities.

[0076] In some embodiments of any of the aforementioned 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 artery stenosis, or parasitic infection.

[0077] In some embodiments of any of the aforementioned aspects, the PH is other PH. In some embodiments, the other PH is associated with a hematologic 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 disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0078] In some embodiments of any of the preceding aspects, the method reduces the frequency or severity of one or more symptoms of PH (e.g., reduces the frequency or severity of one or more of shortness of breath (dyspnea), fatigue, swelling (e.g., edema) of the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, bluish color to the lips or skin (cyanosis), dizziness, or syncope).

[0079] In some embodiments of any of the preceding aspects, the method reduces pulmonary vascular remodeling.

[0080] In some embodiments of any of the preceding aspects, the method reduces vascular remodeling in the heart.

[0081] In some embodiments of any of the preceding aspects, the method reduces right ventricular hypertrophy.

[0082] In some embodiments of any of the aforementioned aspects, the method reduces pulmonary vascular resistance (eg, reduces pulmonary vascular resistance as compared to a measurement taken before treatment).

[0083] In some embodiments of any of the aforementioned aspects, the method improves performance on a 6-minute walk test (eg, improves performance compared to a measurement taken before treatment).

[0084] In some embodiments of any of the aforementioned aspects, the method reduces or inhibits binding of activin and / or myostatin to their endogenous receptors.

[0085] In some embodiments of any of the aforementioned aspects, the amount of the polypeptide, nucleic acid, vector, or pharmaceutical composition administered is sufficient to reduce fibrosis, prevent the development of fibrosis, delay or attenuate the development 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 reduce or inhibit binding of activin and / or myostatin to their endogenous receptors.

[0086] In some embodiments of any of the foregoing aspects, the amount of the polypeptide, nucleic acid, vector, or pharmaceutical composition administered is 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 blood transfusions, treat anemia, affect myostatin, activin, and / or BMP9 signaling in a subject, or reduce or inhibit binding of activin and / or myostatin to their endogenous receptors.

[0087] In some embodiments of any of the foregoing aspects, the amount of the polypeptide, nucleic acid, vector, or pharmaceutical composition administered is 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 development of PH, slow or inhibit the progression of PH, treat PH, reduce pulmonary vascular remodeling, reduce vascular remodeling in the heart, reduce right ventricular hypertrophy, reduce pulmonary vascular resistance, improve performance on a 6-minute walk test, affect myostatin, activin, and / or BMP9 signaling in a subject, or reduce or inhibit the 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.

[0088] In some embodiments of any of the aforementioned aspects, the method does not cause vascular complications in the subject. In some embodiments, the method does not increase vascular permeability or leakage.

[0089] In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 69. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 58. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 6. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 38. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 41. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 44. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 70. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 71. In some embodiments of any of the preceding aspects, the variant has a sequence of SEQ ID NO: 72. In some embodiments of any of the preceding aspects, 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 has a sequence at position X. 17 In some embodiments of any of the preceding aspects, a variant having a 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 26In some embodiments of any of the preceding aspects, the variant of 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 preceding aspects, the method comprises administering to the subject a therapeutically effective amount of a 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). 17 There is amino acid K at position X 23 、X 24 、X 25 and X 26 The invention relates to a pharmaceutical composition comprising a variant of a cytokine receptor antagonist (e.g., a cytokine receptor antagonist having the amino acid sequence TEEN or TKEN and / or a C-terminal extension) to increase, decrease, 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, affect muscle growth in a subject (e.g., a subject having fibrosis, low red blood cell levels, or pH, or at risk of developing fibrosis, low red blood cell levels, or pH). In some embodiments, the present invention relates to an agent that reduces or inhibits myostatin, activin and / or BMP9 signaling (e.g., reduces or inhibits myostatin, activin and / or BMP9 and their endogenous receptors), increases red blood cell levels in a subject (e.g., a subject having anemia or blood loss or at risk of developing anemia or blood loss), increases red blood cell formation in a subject (e.g., a subject having anemia or blood loss or at risk of developing anemia or blood loss), treats a subject having anemia or at risk of developing anemia, prevents PH, reduces the risk of developing PH, slows or inhibits the progression of PH, or treats a subject having PH or at risk of developing PH.

[0090] definition

[0091] As used herein, the term "extracellular activin receptor type IIA (ActRIIA) variant" refers to a peptide comprising the soluble, extracellular portion of the single transmembrane receptor ActRIIA having at least one amino acid substitution relative to wild-type extracellular ActRIIA (e.g., the bold portion of the sequence of SEQ ID NO: 75 shown below) or an extracellular ActRIIA having any of the sequences of SEQ ID NOs: 76-96. The sequence of the wild-type, human ActRIIA precursor protein is shown below (SEQ ID NO: 75), with the signal peptide in italics and the extracellular portion in bold.

[0092] Wild-type, human ActRIIA precursor protein (SEQ ID NO: 75):

[0093]

[0094] The extracellular ActRIIA variant may have the sequence of any one of SEQ ID NOs: 1-72. In specific 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 to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73).

[0095] As used herein, the term "extracellular ActRIIB variant" refers to a peptide comprising a soluble, extracellular portion of a single transmembrane receptor ActRIIB that has at least one amino acid substitution relative to wild-type extracellular ActRIIB (e.g., the sequence of SEQ ID NO: 74). The extracellular ActRIIB variant may have the sequence of SEQ ID NO: 149 as shown below:

[0096] Extracellular ActRIIB variant (SEQ ID NO: 149):

[0097]

[0098] As used herein, the term "linker" refers to a connection between two elements (e.g., a peptide or protein domain). The polypeptides described herein can include an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a portion. The portion can increase stability or improve the pharmacokinetic properties of the polypeptide. The portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) can be fused to the polypeptide by means of a linker. The linker can be a covalent bond or a spacer. The term "bond" refers to a chemical bond, such as an amide bond or a disulfide bond, or any type of bond produced by a chemical reaction (e.g., chemical conjugation). The term "spacer" refers to a portion (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence) that exists between two elements (e.g., peptide or protein domains) to provide space and / or flexibility between the two elements. The amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to the spacer's peptide via the polypeptide backbone). For example, the formation of a disulfide bond between the two hinge regions that form the Fc domain is not considered a linker.

[0099] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. H 2 domains and C H The human Fc domain of the three domains has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97% or 100% sequence identity). The Fc domain monomer comprises the second and third antibody constant domains (C H 2 and C H 3). In some embodiments, the Fc domain monomer further comprises a hinge domain. The Fc domain does not comprise any portion of an immunoglobulin capable of acting as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are connected by two C H 3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. In some embodiments, the Fc domain can be mutated to lack effector function, which is a typical "death Fc domain". In certain embodiments, each Fc domain monomer in the Fc domain comprises C H2. Amino acid substitutions in the antibody constant domain that reduce the interaction or binding between the Fc domain and Fcγ receptors. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0100] As used herein, the term "albumin-binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has an affinity for and binds to serum albumin. The albumin-binding peptide can be of different origins (e.g., human, mouse, or rat). In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0101] As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix or a fragment thereof that binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain of amino acids 610-702 having the sequence of UniProt ID NO: P02751 (SEQ ID NO: 153). In other embodiments, the fibronectin domain is an adnectin protein.

[0102] As used herein, the term "human serum albumin" refers to albumin present in human plasma. Human serum albumin is the most abundant protein in blood. It constitutes about half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0103] As used herein, the term "fusion" is used to describe the combination or attachment of two or more elements, components, or protein domains (e.g., peptides or polypeptides) by means including chemical conjugation, recombinant means, and chemical bonds (e.g., amide bonds). For example, two single peptides in a tandem series can be fused to form a continuous protein structure, such as a polypeptide, by chemical conjugation, chemical bonds, peptide linkers, or any other means of covalent attachment. In some embodiments of the polypeptides described herein, an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having 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 portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., the sequence of SEQ ID NO: 151), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide (e.g., the sequence of SEQ ID NO: 152), a fibronectin domain (e.g., the sequence of SEQ ID NO: 153), or human serum albumin (e.g., the sequence of SEQ ID NO: 154)) by means of a linker in a tandem series. 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 dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by means of a peptide linker, wherein the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIA variant by a chemical bond (e.g., a peptide bond), and the C-terminus of the peptide linker is fused to the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by a chemical bond (e.g., a peptide bond).

[0104] 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 1-6 amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids). Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 155) (a six amino acid C-terminal extension). Any amino acid sequence that does not destroy the activity of the polypeptide can be used. SEQ ID NO: 71 (which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NP) and SEQ ID NO: 72 (which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NPVTPK) represent two possible ways in which a polypeptide of the invention can be modified to include a C-terminal extension.

[0105] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence (e.g., an extracellular ActRIIA variant) that are identical to the amino acid (or nucleic acid) residues of a reference sequence (e.g., wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73)), after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and nonhomologous sequences can be ignored for comparison purposes). Alignment for the purpose of determining percent identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which may alternatively be expressed as a given candidate sequence having or comprising a particular percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0106] 100 x (A / B score)

[0107] wherein A is the number of identical amino acid (or nucleic acid) residues scored in an alignment of the candidate sequence and the reference sequence, and wherein B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence is not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0108] In a specific embodiment, alignment of a reference sequence for comparison to a candidate sequence can show that the candidate sequence exhibits 50% to 100% identity over the full length of the candidate sequence or a selected portion of contiguous 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. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0109] As used herein, the term "serum half-life" refers to the time required for the plasma concentration of a protein in a subject to be reduced by half in the context of administering a therapeutic protein to a subject. Proteins can be redistributed or cleared from the bloodstream, or, for example, degraded by proteolysis. As described herein, polypeptides comprising extracellular ActRIIA variants (e.g., extracellular ActRIIA variants having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) exhibit a serum half-life of 7 days in humans.

[0110] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Typically, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (such as an extracellular ActRIIA variant and BMP9 or activin A). Unless otherwise indicated, binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is typically measured by the dissociation constant (K D ) or affinity constant (K A Two molecules with low binding affinity for each other typically associate slowly, tend to dissociate easily, and exhibit a large K D Two molecules with high affinity for each other generally bind easily, tend to remain bound longer, and exhibit a small K D The K of two interacting molecules can be determined using methods and techniques well known in the art, such as surface plasmon resonance. D .K D Calculated as koff / k on ratio.

[0111] As used herein, the phrase "affecting myostatin, activin and / or BMP9 signaling" refers to altering the binding of myostatin, activin and / or BMP9 to its receptors, e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA). In some embodiments, a polypeptide comprising an extracellular ActRIIA variant as described herein reduces or inhibits the binding of myostatin, activin and / or BMP9 to its receptors, e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA). As described herein, a polypeptide of the invention comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can have a weak binding affinity for BMP9 (e.g., a K of 200 pM or greater). D ).

[0112] As used herein, the terms "increase" and "decrease" refer to modulation of a function, expression, or activity that results in a greater or lesser amount of a metric, respectively, relative to a reference. For example, following administration of a polypeptide of the invention, including an extracellular ActRIIA variant, as described herein, the amount of a marker as measured (e.g., red blood cell count) can be increased or decreased relative to the amount of the marker prior to administration. Typically, the metric is measured at a time after administration at which the effect has been achieved, such as at least one week, one month, three months, or six months after the treatment regimen has begun.

[0113] As used herein, the term "endogenous" describes 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, such as a human cell, e.g., a human hair cell).

[0114] As used herein, the term "fibrosis" refers to a pathological process in which excessive formation of fibrous connective tissue occurs. Fibrosis is characterized by accumulation of fibroblasts and deposition of collagen that exceeds normal deposition in any particular tissue. In response to inflammation or tissue damage, nearby fibroblasts can migrate into the wound, proliferate, and produce large amounts of collagen extracellular matrix. When fibrosis occurs in response to damage, the term "scar" can be used synonymously. Fibrosis can occur in many tissues of the body, including, for example, the lungs, skin, liver, kidneys, heart, eyes, tendons, cartilage, pancreatic tissue, uterine tissue, neural tissue, testicles, ovaries, adrenal glands, arteries, veins, colon, small and large intestine, bile duct, and intestines.

[0115] As used herein, the term "pulmonary hypertension" or "PH" refers to a disease characterized by increased blood pressure between the lungs and the heart, which can include increased blood pressure in the pulmonary arteries (pulmonary hypertension), pulmonary veins, or pulmonary capillaries. Pulmonary hypertension can have a variety of symptoms, including shortness of breath (dyspnea), fatigue, swelling (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, a bluish discoloration of the lips or skin (cyanosis), dizziness, or syncope. PH is also characterized by reduced exercise tolerance and can lead to heart failure.

[0116] As used herein, the term "pulmonary arterial hypertension" or "PAH" refers to a form of pulmonary hypertension characterized by stenosis or obstruction in the small pulmonary arteries, often caused by scarring, and an increase in pulmonary artery blood pressure. PAH is also known as WHO Class I PH. PAH can be diagnosed based on an increase in blood pressure in the pulmonary artery mean pulmonary artery pressure above 25 mmHg at rest, accompanied by a normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, syncope, and other symptoms, all of which are exacerbated by exertion. PAH can be a serious disease with significantly reduced exercise tolerance and heart failure. The two main types of PAH include idiopathic PAH (e.g., PAH in which no predisposing factor has been identified) and heritable PAH (e.g., PAH associated with mutations in BMPR2, ALK1, SMAD9, caveolin 1, KCNK3, or EIF2AK4). In 70% of familial PAH cases, the mutation is located in the BMPR2 gene. Risk factors for the development of PAH include a family history of PAH, drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus).

[0117] As used herein, the terms "venopulmonary hypertension" and "venous PH" refer to a form of pulmonary hypertension secondary to left heart disease. Venous PH is also known as WHO Class II PH. Venous PH may 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 or aortic valve disease), congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis.

[0118] As used herein, the terms "hypoxic pulmonary hypertension" and "hypoxic PH" refer to a form of pulmonary hypertension due to lung disease or chronic hypoxia. This form of PH is also known as WHO Class III PH. Hypoxic PH may 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, long-term exposure to high altitude, or developmental abnormalities.

[0119] 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., blood clot). Thromboembolic PH is also known as WHO Class IV PH. Thromboembolic PH may be associated with or caused by chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection).

[0120] As used herein, the terms "other pulmonary hypertension" and "other PH" refer to a form of pulmonary hypertension with an unclear or multifactorial mechanism. This form of PH is classified as WHO class V PH. Other PH may be associated with or caused by hematologic 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 tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0121] As used herein, the term "red blood cell levels" refers to clinically observable measurements such as hematocrit, red blood cell count, and hemoglobin measurements. As used herein, the terms "increase red blood cell levels" and "promote red blood cell formation" refer to clinically observable measurements such as hematocrit, red blood cell count, and hemoglobin measurements, and are expected 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 the subject's age and sex.

[0122] As used herein, the term "erythrocyte mass" refers to the total number of red blood cells in circulation, which is typically reduced in anemia.

[0123] As used herein, the terms "erythropoiesis" and "erythropoiesis" 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.

[0124] As used herein, the term "anemia" refers to any abnormality in hemoglobin or red blood cells that results in a decrease in oxygen levels in the blood. Anemia can be associated with abnormal production, processing, or properties 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 relative to normal blood levels.

[0125] As used herein, the term "vascular complication" refers to a vascular disorder or any damage to a blood vessel, such as damage to the vessel wall. Damage to the vessel wall can cause an increase in vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the vessel wall to allow small molecules, proteins, and cells to flow into and out of the vessel. Increased vascular permeability or leakage may be caused by an increase in the gaps between the endothelial cells lining the vessel wall (e.g., an increase in the size and / or number of the gaps) and / or thinning of the vessel wall.

[0126] As used herein, the term "polypeptide" describes a single polymer in which the monomers are amino acid residues covalently joined together by amide bonds. Polypeptide is intended to encompass any amino acid sequence that is naturally occurring, recombinant, or synthetically produced.

[0127] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules (such as proteins or nucleic acids). Two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, if two Fc domain monomers contain the same sequence, the Fc domain can be a homodimer of two Fc domain monomers. In another example, a polypeptide comprising an extracellular ActRIIA variant fused to an Fc domain monomer as described herein can form a homodimer through the interaction of two Fc domain monomers, which form an Fc domain in a homodimer.

[0128] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules (such as proteins or nucleic acids). Two monomers can form a heterodimer 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 of which is fused to a different ActRIIA variant), wherein the Fc domain monomers form an Fc domain in the heterodimer.

[0129] As used herein, the term "host cell" refers to a vehicle comprising the necessary cellular components (e.g., organelles) required for expressing a protein from its corresponding nucleic acid. Nucleic acids are typically contained in nucleic acid vectors that can be introduced into host cells by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells can be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells).

[0130] 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 the polypeptide, nucleic acid or vector of the invention, effective to achieve the 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 or other PH). Specifically, a therapeutically effective amount of a polypeptide, nucleic acid or vector avoids adverse side effects.

[0131] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or drug formulation comprising an active ingredient and excipients and diluents that render the active ingredient suitable for a method of administration. The pharmaceutical compositions of the present invention comprise pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition can be in the form of tablets or capsules for oral administration, or in aqueous form for intravenous or subcutaneous administration.

[0132] 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 adequate pharmaceutical stability for the polypeptide comprising the extracellular ActRIIA variant, the 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, for intravenous administration, aqueous carriers are typically used; for oral administration, solid carriers are preferred.

[0133] 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. Typically, treatment of anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) occurs after the subject has developed anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) and / or has been diagnosed with 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 anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). A subject may display signs or mild symptoms that are judged by a physician to be indicative of or risk factors for developing anemia, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), have 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), be undergoing treatment (e.g., surgery, chemotherapy, or radiation) that may cause anemia or fibrosis, or have 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 have not yet developed the disease or condition.

[0134] 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.

[0135] Description of the drawings

[0136] Figure 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIA and ActRIIB and the amino acid substitutions in ActRIIA variants.

[0137] Figure 2 is a series of graphs showing that treatment of elderly mdx mice with ActRIIA / B-Fc (A / B, SEQ ID NO: 69 fused to the Fc domain) attenuates the development of fibrosis.

[0138] Figure 3is a graph showing that treatment of non-human primates with ActRIIA / B-Fc (SEQ ID NO: 69 fused to the Fc domain) increases red blood cell mass. Hct – hematocrit, Hgb – hemoglobin, RBC – red blood cell count. Detailed Description of the Invention

[0140] The present invention features polypeptides comprising extracellular activin receptor type IIA (ActRIIA) variants. In some embodiments, the polypeptides of the invention comprise an extracellular ActRIIA variant fused to a portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIA variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The ActRIIA variants described herein have weak or no binding affinity for bone morphogenetic protein 9 (BMP9) compared to activin and myostatin. The present invention also includes methods of treating or preventing fibrosis by administering to a subject a polypeptide comprising an extracellular ActRIIA variant described herein, methods of treating or preventing low red blood cell levels (e.g., anemia or blood loss) by increasing red blood cell levels (e.g., red blood cell count, hemoglobin level, or hematocrit, e.g., increasing red blood cell mass) or red blood cell production, methods of treating or preventing pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), or methods of affecting myostatin, activin, and / or BMP9 signaling in a subject.

[0141] I. Extracellular activin receptor type IIA (ActRIIA) variants

[0142] Activin type II receptors are single transmembrane domain receptors that regulate the signaling of ligands in the transforming growth factor beta (TGF-β) superfamily. Ligands in the TGF-β superfamily are involved in many physiological processes, such as muscle growth, angiogenesis, cell differentiation, homeostasis, and osteogenesis. Examples of ligands in the TGF-β superfamily include, for example, activins, inhibins, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin, and GDF11), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activins have been shown to regulate fibrosis. For example, mice lacking myostatin show a decrease in muscle fibrosis, while injection of myostatin-coated beads induces muscle fibrosis in mice. Mice that overexpress activin subunits also show fibrosis, which results in the production of diffusible activin A. Activin A elevation has also been observed in clinical and experimental pulmonary hypertension. Another activin type II receptor ligand, GDF11, has been shown to be overexpressed in a mouse model of beta-thalassemia and is associated with ineffective red blood cell production. Therefore, reducing or inhibiting signaling through type II activin receptors (e.g., signaling mediated by myostatin, activin, and / or BMP11) may be useful in treating fibrosis, conditions involving low red blood cell levels (e.g., anemia), and pulmonary hypertension (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).

[0143] There are two types of activin type II receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds to ActRIIB with an affinity approximately 300-fold higher than that of ActRIIA (see, e.g., Townson et al., J. Biol. Chem.287:27313, 2012). ActRIIA is known to have a longer half-life compared to ActRIIB. The present invention describes extracellular ActRIIA variants constructed by introducing amino acid residues of ActRIIB into ActRIIA, the purpose of which is to confer the physiological properties conferred by ActRIIB while also maintaining the beneficial physiological and pharmacokinetic properties of ActRIIA. For example, the optimal peptides reduce fibrosis, treat PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH or other PH), and / or increase red blood cell levels (e.g., increase 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, which allows them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling. These variants can be used to treat conditions in which activin receptor signaling is elevated, such as fibrosis, PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), and anemia, resulting in a decrease in fibrosis (e.g., a decrease in fibrosis, a slowing or halting of the progression of fibrosis, an improvement in existing fibrosis, or a reversal of existing fibrosis), an increase in red blood cell levels (e.g., an increase in hemoglobin levels, hematocrit, or red blood cell count, e.g., an increase in red blood cell production and / or red blood cell mass), or a decrease in the 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 the extracellular ActRIIA variant to reduce or eliminate the binding affinity of the variant to BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIA and ActRIIB are shown below.

[0144] Human ActRIIA, extracellular portion (SEQ ID NO: 73):

[0145]

[0146] Human ActRIIB, extracellular portion (SEQ ID NO: 74):

[0147]

[0148] The polypeptides described herein include extracellular ActRIIA variants having at least one amino acid substitution relative to wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73 or extracellular ActRIIA having any one of the sequences of SEQ ID NOs: 76-96. Possible amino acid substitutions at 27 different positions can be introduced into extracellular ActRIIA variants (Table 1). In some embodiments, the extracellular ActRIIA variants can have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97% or more) amino acid sequence identity to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73). The extracellular ActRIIA variant can have one or more (e.g., 1-27, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) amino acid substitutions relative to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73). In some embodiments, the extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of SEQ ID NO: 1) can comprise amino acid substitutions at all 27 positions as listed in Table 1. In some embodiments, the extracellular ActRIIA variants can comprise amino acid substitutions at a number of positions, for example, at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 of the 27 positions as listed in Table 1.

[0149] Amino acid substitutions can worsen or improve the activity and / or binding affinity of the ActRIIA variants of the present invention. To maintain polypeptide function, it is important to retain position X in the sequences shown in Tables 1 and 2 (SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). 17 Substitution at this position may result in loss of activity. For example,

[0150] The ActRIIA variant of GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reduced in vivo activity, indicating that it cannot tolerate alanine (A) substitution at position X. 17Thus, the ActRIIA variants of the present invention, including those in Tables 1 and 2 (e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), have a lysine (K) at position X. 17 The amino acid K is retained.

[0151] The ActRIIA variants of the present invention preferably have reduced, weak, or substantially no binding to BMP9. 23 、X 24 、X 25 and X 26 and maintaining the amino acid K at position X24 and having an amino acid residue at position X24. 23 、X 24 、X 25 and X 26 In variants of the amino acid sequence TKEN at TEEN, BMP9 binding is reduced (e.g., reduced compared to wild-type ActRIIA). The sequences TEEN and TKEN can be used interchangeably in ActRIIA variants of the invention (e.g., variants in Tables 1 and 2, e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to provide reduced BMP9 binding.

[0152] The ActRIIA variants of the present invention may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension can add one to six additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) at the C-terminus to any of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). One potential C-terminal extension that can be included in the ActRIIA variants of the present invention 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). Another exemplary C-terminal extension that can be included in the ActRIIA variants of the present invention is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, a sequence comprising the C-terminal extension NPVTPK is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK).

[0153] Table 1. Amino acid substitutions in extracellular ActRIIA variants having the sequence of any one of SEQ ID NOs: 1-5

[0154]

[0155] In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 2, X3 is E, X6 is R, X 11 It's D, X 12 It's K, X 13 It's R, X 16 Is K or R, X 17 It's K, X 19 It's W, X 20 It's L, X 21 is D, and X 22 Is I or F. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X 17 Is K. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NOs: 1-3, X 17 It's K, X 23 It's T, X 24 It's E, X 25 is E, and X 26 Is N. In some embodiments of the extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-5, X 17 It's K, X 23 It's T, X 24 It's K, X 25 is E, and X 26 It's N.

[0156] In some embodiments, a polypeptide described herein comprises an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 6-72 (Table 2).

[0157] Table 2. Extracellular ActRIIA variants having sequences of SEQ ID NOs: 6-72

[0158]

[0159]

[0160]

[0161]

[0162] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) has position X 17 Amino acid K at position X 17 The amino acid residues at the end of the sequence may result in decreased activity.

[0163] The ActRIIA variant of GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reduced in vivo activity, indicating that it cannot tolerate the substitution of A for position X. 17 K at.

[0164] In some embodiments, comprising a 23 、X 24 、X 25 and X 26 A polypeptide of the invention having an extracellular ActRIIA variant of the sequence TEEN at position (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may substitute position X with amino acid K. 24 In some embodiments, the amino acid E at position X 23 、X 24 、X 25 and X 26 The polypeptides of the invention having an extracellular ActRIIA variant of the sequence TKEN at position (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can substitute amino acid E for position X 24 The amino acid K at position X 23 、X 24 、X 25 and X 26 A polypeptide having the sequence TEEN or TKEN at the position of ActRIIA has reduced or weak binding to BMP9 (eg, the binding to BMP9 is reduced compared to the BMP9 binding of wild-type ActRIIA).

[0165] 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)) can further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). In some embodiments, the C-terminal extension is the amino acid sequence NP. For example, the sequence comprising the C-terminal extension 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, the 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 add one to six additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus.

[0166] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIA variant may further comprise a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) that is fused to the N- 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 may form a dimer (e.g., a homodimer or heterodimer) through interaction between two Fc domain monomers that combine to form the Fc domain in the dimer.

[0167] In some embodiments, the extracellular ActRIIA variants described herein do not have the sequence of any of SEQ ID NOs: 76-96 shown in Table 3 below.

[0168] Table 3. Excluded extracellular ActRIIA variants.

[0169]

[0170]

[0171]

[0172] Furthermore, in some embodiments, the polypeptides described herein have a serum half-life of at least 7 days in humans. DBinds to bone morphogenetic protein 9 (BMP9). The polypeptide can bind to bone morphogenetic protein 9 (BMP9) with a K of 10 pM or higher. D Binds to activin A. In some embodiments, the polypeptide does not bind to BMP9 or activin A. In some embodiments, the polypeptide binds to activin and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., BMP9 binding is reduced compared to BMP9 binding of wild-type ActRIIA). In some embodiments, the polypeptide with reduced or weak binding to BMP9 is at position X 23 、X 24 、X 25 and X 26 The sequence TEEN or TKEN is present at the position.

[0173] Additionally, in some embodiments, the polypeptide can be expressed as a peptide with a K of about 200 pM or greater. D (e.g., a K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or more D , for example, a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or more D , for example, a K of about 200 pM to about 50 nM D ) binds to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9. In some embodiments, the polypeptide can bind to human BMP9 with a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin A. In some embodiments, the polypeptide can bind to human activin A with a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin B. The polypeptide may also bind to human activin B with a K of about 5 pM or higher. D(e.g., a K of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or more) D ) binds to growth and differentiation factor 11 (GDF-11).

[0174] II. Fc domain

[0175] 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 increase 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 heterodimer) by interaction between two Fc domain monomers, which form an Fc domain in the dimer. As is conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain comprises two Fc domain monomers, which are bound by a C H3 antibody constant domains and dimerize. Wild-type Fc domains form the minimum structure that binds to Fc receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, which is a typical "dead" Fc domain. For example, the Fc domain can include specific amino acid substitutions known to minimize the interaction between the Fc domain and Fcγ receptors. In some embodiments, the Fc domain is from an IgG1 antibody and includes the amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is from an IgG1 antibody and includes the amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). By aligning the homology regions of the antibody sequence with the "standard" Kabat numbering sequence, the Kabat numbering of the amino acid residues of a given antibody can be determined. In addition, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a dimer of a polypeptide comprising an extracellular ActRIIA variant fused to an Fc domain monomer can be modified to reduce the interaction or binding between the Fc domain and Fcγ receptors. The sequence of the Fc domain monomer that can be fused to the extracellular ActRIIA variant is shown below (SEQ ID NO: 97):

[0176]

[0177] In some embodiments, the Fc domain is derived from an IgG1 antibody and comprises amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO: 97. In some embodiments, the Fc domain is derived from an IgG1 antibody and comprises amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO: 97. In some embodiments, the extracellular ActRIIA variants described herein (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and the Fc domain monomer. The Fc domain monomer can be fused to the N- or C-terminus (e.g., the C-terminus) of the extracellular ActRIIA variant.

[0178] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to an Fc domain. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain contains a hinge domain. The Fc domain can be an Fc domain of an immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0179] Methods for engineering Fc domains with reduced dimerization are known in the art. In some embodiments, one or more amino acids with large side chains (e.g., tyrosine or tryptophan) can be introduced into the C H 3-C H 3 dimer interface to hinder dimer formation due to steric clashes. In other embodiments, one or more amino acids with small side chains (e.g., alanine, valine, or threonine) can be introduced into the C H 3-C H 3 dimer interface to remove favorable interactions. H Methods for introducing amino acids with large or small side chains into the 3 domain are described, for example, by Ying et al. ( J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication No. 2006 / 0074225, U.S. Patent Nos. 8,216,805 and 5,731,168, Ridgway et al. ( Protein Eng. 9:617-612, 1996), Atwell et al. J Mol Biol. 270:26-35, 1997), and Merchant et al. ( Nat Biotechnol. 16:677-681, 1998), all of which are incorporated herein by reference in their entirety.

[0180] In yet other embodiments, the C segment between the two Fc domains is H 3-C H 3 interface C H One or more amino acid residues in the C-terminal domain are replaced 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) such that the interaction becomes electrostatically unfavorable, depending on the specific charged amino acid introduced. H Methods for introducing charged amino acids into the 3 domain to disfavor or prevent dimer formation are described, for example, by Ying et al. ( J Biol Chem . 287:19399-19408, 2012), U.S. Patent Publication Nos. 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0181] In some embodiments of the invention, the Fc domain comprises one or more of the following amino acid substitutions relative to the sequence of human IgG1: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, In one embodiment, the Fc domain comprises the amino acid substitution T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I. In a specific embodiment, the Fc domain comprises the amino acid substitution T366W relative to the sequence of human IgG1. The sequence of the wild-type Fc domain is shown in SEQ ID NO: 151.

[0182] III. Albumin-binding peptides

[0183] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to a serum protein binding peptide. Binding to a serum protein peptide may improve the pharmacokinetics of protein drugs.

[0184] As an example, albumin binding peptides useful in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0185] In the present invention, an albumin-binding peptide can be linked to the N- or C-terminus (e.g., the C-terminus) of an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular ActRIIA variant. In some embodiments, the albumin-binding peptide is linked directly or via a linker to the N- or C-terminus of the extracellular ActRIIA variant.

[0186] In some embodiments, the extracellular ActRIIA variants 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 NO: 152) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and the albumin-binding peptide. Without being bound by theory, it is contemplated that the inclusion of an albumin-binding peptide in the extracellular ActRIIA variants described herein can result in prolonged retention of the therapeutic protein through its binding to serum albumin.

[0187] IV. Fibronectin Domain

[0188] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to a fibronectin domain. Binding to the fibronectin domain may improve the pharmacokinetics of protein drugs.

[0189] The fibronectin domain is a high molecular weight glycoprotein of the extracellular matrix or a fragment thereof that binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, the fibronectin domain is connected to the N- or C-terminus (e.g., C-terminus) of an extracellular ActRIIA variant described herein (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular ActRIIA variant. The fibronectin domain can be connected directly or via a linker to the N- or C-terminus of the extracellular ActRIIA variant.

[0190] As an example, fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain 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.

[0191] In some embodiments, the extracellular ActRIIA variants 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 NO: 153) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and the fibronectin domain. Without being bound by theory, it is contemplated that the inclusion of a fibronectin domain in the extracellular ActRIIA variants described herein can result in prolonged retention of the therapeutic protein through its binding to integrins and extracellular matrix components (such as collagen and fibrin).

[0192] V. Serum albumin

[0193] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIA variant fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of protein drugs.

[0194] Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and accounts for about half of serum proteins. It is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure required for the correct distribution of body fluids between blood vessels and body tissues. In a preferred embodiment, serum albumin is human serum albumin. In some embodiments of the present invention, human serum albumin is linked to the N- or C-terminus (e.g., C-terminus) of 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)) to increase the serum half-life of the extracellular ActRIIA variant. Human serum albumin can be linked directly or via a linker to the N- or C-terminus of the extracellular ActRIIA variant.

[0195] As an 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).

[0196] In some embodiments, the extracellular ActRIIA variants 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 human serum albumin (e.g., SEQ ID NO: 154) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIA variant and the human serum albumin. Without being bound by theory, it is expected that the inclusion of human serum albumin in the extracellular ActRIIA variants described herein can result in prolonged retention of the therapeutic protein.

[0197] VI. Connectors

[0198] The polypeptides described herein may comprise an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety by means of a linker. In some embodiments, the moiety increases the stability of the polypeptide. Exemplary moieties include an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin. In the present invention, the linker between a portion (e.g., an Fc domain monomer (e.g., sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., SEQ ID NO: 151), an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide (e.g., SEQ ID NO: 152), a fibronectin domain (e.g., SEQ ID NO: 153, or human serum albumin (e.g., SEQ ID NO: 154)) and an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be an amino acid spacer comprising 1-200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the spacer region can contain a motif, such as a multiple or repeated motif, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO:98), GGGS (SEQ ID NO:99), GGGG (SEQ ID NO:100), GGGGA (SEQ ID NO:101), GGGGS (SEQ ID NO:102), GGGGG (SEQ ID NO:103), GGAG (SEQ ID NO:104), GGSG (SEQ ID NO:105), AGGG (SEQ ID NO:106), or SGGG (SEQ ID NO:107).In some embodiments, the spacer region can contain 2 to 12 amino acids, including GA or GS, such as the motif of GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer region can contain 3 to 12 amino acids, including the motifs of GGA or GGS, such as GGA, GGS, GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). In still other embodiments, the spacer can contain 4 to 12 amino acids, including the motifs of GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), e.g., GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In some embodiments, the spacer can contain the motifs of GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128), and GGGGSGGGGSGGGGS (SEQ ID NO: 129).In some embodiments of the invention, the linker between the portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) and 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)) can be GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

[0199] In some embodiments, the spacer can also contain amino acids other than glycine, alanine, and serine, such as AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), In some embodiments, the spacer may contain a motif, such as multiple or repeated motifs, of EAAAK (SEQ ID NO: 147). In some embodiments, the spacer may contain a proline-rich sequence, such as (XP) n ), such as a multiple or repeating motif, wherein X can be any amino acid (e.g., A, K, or E) and n is 1-5, and PAPAP (SEQ ID NO: 148).

[0200] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0201] VII. Vectors, Host Cells, and Protein Production

[0202] The polypeptide of the present invention can be produced from a host cell. A host cell refers to a vehicle comprising essential cellular components (e.g., organelles) required for expressing the polypeptide described herein and the fusion polypeptide from its corresponding nucleic acid. Nucleic acid can be included in a nucleic acid vector, which can be introduced into a host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, injection, etc.). The selection of nucleic acid vectors depends in part on the host cell to be used. Typically, preferred host cells are eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origins.

[0203] Nucleic acid vector construction and host cells

[0204] The nucleic acid sequence encoding the amino acid sequence of the polypeptide of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptide of the present invention can be obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques in the art (e.g., QuikChange TM Mutagenesis) mutates a nucleic acid molecule encoding wild-type extracellular ActRIIA to include a specific amino acid substitution. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0205] The nucleotide sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing nucleic acid molecules in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for purposes of the present invention. Every kind of vector can include various components that can be adjusted and optimized for compatibility with a specific host cell. For example, vector components can include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding a target protein, and a transcription termination sequence.

[0206] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells. In some embodiments, E. coli cells can also be used as host cells of the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC ® 31,446), Escherichia coli λ1776 (ATCC ® 31,537, Escherichia coli BL21 (DE3) (ATCC ® BAA-1025), and Escherichia coli RV308 (ATCC ® 31,608). Different host cells have characteristics and specific mechanisms for post-transcriptional processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed polypeptide. The above-mentioned expression vectors can be introduced into appropriate host cells using conventional techniques in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). Once the vector is introduced into the host cell for protein production, the host cell is cultured in a conventional nutrient medium appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequence. Methods for expressing therapeutic proteins are known in the art, see, for example, Paulina Balbas, Argelia Lorence (ed.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology) , HumanaPress; 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.

[0207] Protein production, recovery, and purification

[0208] The host cell for producing the polypeptide of the present invention can be grown in the culture medium of the host cell selected as known in the art and suitable for cultivating. The example of suitable culture medium for mammalian host cells includes minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Expi293™ expression culture medium, DMEM and RPMI-1640 supplemented with fetal bovine serum (FBS). The example of suitable culture medium for bacterial host cells includes Luria broth (LB) plus necessary supplements, such as selection agents, for example ampicillin. The host cell is cultivated at a suitable temperature such as about 20°C to about 39°C, for example 25°C to about 37°C, preferably 37°C, and CO2 levels, such as 5 to 10%. The pH of the culture medium is generally about 6.8 to 7.4, for example 7.0, which depends primarily on the host organism. If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter.

[0209] In some embodiments, depending on the expression vector and host cell used, the expressed protein can be secreted into the cell culture medium from the host cell (for example, a mammalian host cell). Protein recovery can involve filtering the cell culture medium to remove cell debris. Protein can be further purified. The polypeptide of the present invention can be purified by any method known in the field of protein purification, for example, by chromatography (for example, ion exchange, affinity and size exclusion column chromatography), centrifugal, differential solubility or by any other standard technique for protein purification. For example, affinity columns, such as protein A columns (for example, POROS protein A chromatography) and chromatographic columns (for example, POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out and dialysis procedures can be used to separate and purify protein.

[0210] In other embodiments, the host cells can be destroyed, for example, by osmotic shock, sonication, or lysis to recover the expressed protein. Once the cells are destroyed, the cell debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be conjugated to a marker sequence (such as a peptide) to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His-tag), which binds to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags that can be used for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 2001). Cell 37:767, 1984).

[0211] Alternatively, the polypeptides of the invention can be produced by cells of a subject (e.g., a human), for example, in the context of gene therapy, by administering a vector (such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxviral vector (e.g., a vaccinia virus vector, such as modified vaccinia virus Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding the polypeptides of the invention. The vector, once within the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), will promote expression of the polypeptide, which is then secreted from the cells. If treatment of the disease or condition is the desired outcome, no further action may be required. If it is desired to collect the protein, blood can be collected from the subject and the protein purified from the blood by methods known in the art.

[0212] VIII. Pharmaceutical Compositions and Formulations

[0213] The invention features 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: 1-72 (e.g., SEQ ID NOs: 6-72))). In some embodiments, a pharmaceutical composition of the invention comprises a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the present invention comprise a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a portion (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the present invention comprising a polypeptide of the present invention can be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions for therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical composition of the present invention comprises a nucleic acid molecule (DNA or RNA, such as mRNA) encoding a polypeptide of the present invention, or a vector containing such a nucleic acid molecule.

[0214] In the pharmaceutical composition, acceptable carrier and excipient are nontoxic to receptors at dosage and concentration used.Acceptable carrier and excipient can include buffers, such as phosphate, citrate, HEPES and TAE, antioxidants, such as ascorbic acid and methionine, preservatives, such as hexamethylammonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol and benzalkonium chloride, protein, such as human serum albumin, gelatin, dextran and immunoglobulin, hydrophilic polymers, such as polyvinylpyrrolidone, amino acids, such as glycine, glutamine, histidine and lysine, and carbohydrates, such as glucose, mannose, sucrose and sorbitol.Pharmaceutical composition of the present invention can be administered parenterally in the form of injectable preparations.Sterile solution or any pharmaceutically acceptable liquid can be used to prepare the pharmaceutical composition for injection as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's modified Eagle's medium (DMEM), α-modified Eagle's 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 ed.) Taylor & Francis Group, CRC Press (2015).

[0215] The pharmaceutical composition of the present invention can be prepared in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules. The pharmaceutical composition of the present invention can also be prepared in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules. Such technology is described in Remington: The Science and Practice of Pharmacy 22nd Edition (2012). The pharmaceutical composition to be used for in vivo administration must be sterile. This can be easily accomplished by filtration through a sterile filtration membrane.

[0216] The pharmaceutical composition of the present invention can also be prepared as a sustained release formulation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide of the present invention. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM Some sustained release formulations enable release of the molecule over several months, for example one to six months, while other formulations release the pharmaceutical compositions of the invention for shorter periods of time, for example days to weeks.

[0217] The pharmaceutical composition can be formed in unit dosage form as needed. The amount of active ingredient (eg, a polypeptide of the present invention) included in the pharmaceutical preparation is such as to provide a suitable dosage within the specified range (eg, a dosage within the range of 0.01-100 mg / kg body weight).

[0218] The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can include a sustained-release matrix wherein embedded in a gene delivery vector. If hydrodynamic injection is used as a delivery method, the pharmaceutical composition containing the nucleic acid molecules encoding polypeptides as described herein or the carrier (for example, viral vector) containing the nucleic acid molecules is delivered quickly intravenously with a large fluid volume. The carrier that can be used as a gene delivery vehicle in vivo includes but is not limited to retroviral vectors, adenoviral vectors, poxvirus vectors (for example, vaccinia virus vectors, such as the vaccinia virus Ankara of modification), adeno-associated virus vectors and alphavirus vectors.

[0219] IX. Route, Dosage, and Administration

[0220] Pharmaceutical compositions comprising the polypeptide of the present invention as therapeutic proteins can be formulated for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical compositions can also be formulated for oral, nasal, spray, aerosol, rectal, or vaginal administration or administered via oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective drug carriers are known in the art. See, for example, ASHP Handbook on Injectable Drugs, Toissel, 18th edition (2014).

[0221] In some embodiments, the pharmaceutical composition comprising a nucleic acid molecule encoding a polypeptide of the present invention or a vector containing such a nucleic acid molecule can be administered by gene delivery. The method of gene delivery is well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified vaccinia virus Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the mRNA molecule encoding a polypeptide of the present invention can be directly administered to a subject.

[0222] In some embodiments of the present invention, the nucleic acid molecules encoding polypeptides as described herein or the carrier containing such nucleic acid molecules can be used to inject platform.In the hydrodynamic injection method, the nucleic acid molecules encoding polypeptides as described herein are placed under the control of the strong promoter in the plasmid (such as viral plasmid) of engineering modification. Often with large fluid volume intravenous rapid delivery plasmid. Hydrodynamic injection uses the controlled fluid dynamic pressure in vein to enhance cell permeability so that the pressure from the rising of rapid injection large fluid volume causes fluid and plasmid to extravasate from vein. The expression of nucleic acid molecules is mainly driven by the liver. In mice, hydrodynamic injection is often carried out by injecting plasmid into the tail vein. In certain embodiments, the mRNA molecules encoding polypeptides as described herein can be used to inject.

[0223] The dosage of the pharmaceutical composition of the present invention depends on factors, including route of administration, disease to be treated and physical characteristics of the subject, such as age, weight, overall health. The pharmaceutical composition of the present invention can include a dosage of a polypeptide of the present invention ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg / kg), and in a more specific embodiment, from about 0.1 to about 30 mg / kg, and in a more specific embodiment, from about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors, such as the degree of the disease and the different parameters of the subject.

[0224] The pharmaceutical composition is administered in a manner compatible with the dosage formulation and in an amount effective for the treatment of the improvement or remedy of the symptoms. The pharmaceutical composition is administered in various dosage forms, such as intravenous dosage forms, subcutaneous dosage forms and oral dosage forms (e.g., ingestible solutions, drug release capsules). Typically, therapeutic proteins are administered at 0.1-100 mg / kg, such as 1-50 mg / kg. The pharmaceutical composition comprising the polypeptide of the present invention can be administered to a subject in need thereof, for example, every day, every week, every two weeks, every month, every two months, every quarter, every two years, once or multiple times per year (e.g., 1-10 times or more), or when medically necessary. In some embodiments, the pharmaceutical composition comprising the polypeptide of the present invention can be administered to a subject in need thereof weekly, every two weeks, every month, every two months or every quarter. Dosage can be provided in a single dose or multiple dose regimen. As the patient's health declines, as medical conditions improve or increase, the opportunity between administrations can be reduced.

[0225] X. Treatment Methods

[0226] The present invention is based on the discovery that amino acid substitutions from the extracellular portion of ActRIIB into the extracellular portion of ActRIIA produce ActRIIA variants with improved properties. ActRIIA variants generated by introducing residues from ActRIIB into ActRIIA retain the beneficial properties of ActRIIA, such as a longer serum half-life and low binding affinity for BMP9, and gain some of the beneficial properties of ActRIIB, such as increased binding to activin A and B (see Table 5 in Example 1). These ActRIIA variant properties create useful therapeutic agents that can compete with endogenous activin receptors for ligand binding. Because ActRIIA variants contain the extracellular portion of the receptor, they will be soluble and able to bind and sequester ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Therefore, extracellular ActRIIA variants can be used to treat diseases or conditions in which elevated activin signaling has been implicated (e.g., associated with increased expression of activin receptors or activin receptor ligands). For example, loss of myostatin has been shown to reduce fibrosis, while increased 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 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).

[0227] The present invention provides compositions and methods of treatment 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 methods of treatment that can be used to increase red blood cell levels (e.g., increase hemoglobin, increase hematocrit, or increase red blood cell count, e.g., increase red blood cell production and / or red blood cell mass) in a subject in need thereof. The present invention further provides compositions and methods of treatment that can be used to treat PH in a subject in need thereof, reduce PH (e.g., reduce symptoms of PH, such as shortness of breath (dyspnea), fatigue, swelling (e.g., edema) of the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, bluish color (cyanosis) of the lips or skin, dizziness or syncope), or slow or stop the progression of PH. The present invention can treat or 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 may have a disease or condition associated with fibrosis (e.g., 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), or 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 angiomatosis, cirrhosis, congenital heart anomalies, 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 hypoxic PH, such as chronic obstructive pulmonary disease (e.g., such as emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, long-term 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, or parasitic infection); or diseases or conditions associated with other PH, such as hematologic 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 tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension (pulmonary hypertension limited to one or more lobes).In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling in a subject having a disease or condition involving fibrosis, low red blood cell levels, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, a polypeptide comprising an extracellular ActRIIA variant described herein reduces or inhibits the binding of myostatin, activin, and / or BMP9 to its endogenous receptors, such as ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA). In some embodiments, affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB and BMPRII (e.g., ActRIIA)) results in a reduction in fibrosis, a reduction in the risk of developing fibrosis, a delay in the development of fibrosis, a reduction (e.g., slowing or inhibition) in the progression of fibrosis, reversal of fibrosis, an increase in red blood cell levels (e.g., an increase in hemoglobin, hematocrit levels 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 (e.g., edema) of the legs, feet, abdomen (ascites) or neck, chest pain or pressure, a rapid pulse or palpitations, a bluish color to the lips or skin (cyanosis), dizziness or syncope), a reduction in the risk of developing PH, a delay in the development of PH and / or a reduction (e.g., slowing or inhibition) in the progression of PH. PH can be PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.

[0228] The compositions and methods described herein can be used to treat and / or prevent (e.g., prevent the development 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 counts), fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, a polypeptide described herein (e.g., comprising an extracellular ActRIIA variant (e.g., having SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 1-73)) can be administered to a subject in need thereof. NO: 6-72), such as an effective amount of an ActRIIA variant) 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). 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) as compared to measurements obtained before treatment. In some embodiments, the subject may have a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss). In some embodiments, the subject may have anemia, A subject may be at risk of developing anemia if he or she has or is at risk of developing anemia or blood loss (e.g., due to another disease or condition, such as chronic kidney disease, rheumatoid arthritis, cancer, or an inflammatory disease (e.g., Crohn's disease, SLE, or ulcerative colitis), or due to medical treatment (e.g., 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 binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in a subject having a disease or condition involving low red blood cell levels (e.g., anemia or blood loss).

[0229] 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 prevent or reduce fibrosis. In some embodiments, a polypeptide described herein can be administered to slow or halt the progression of fibrosis, to reduce the risk of developing fibrosis, to reduce (e.g., reduce the frequency or severity of) one or more symptoms of fibrosis, or to reverse fibrosis. The polypeptides described herein can reduce fibrosis or slow or reverse the progression of fibrosis compared to the progression of fibrosis before treatment or compared to the progression of fibrosis in an untreated subject. In some embodiments, a subject may have fibrosis or be at risk of developing fibrosis (e.g., a 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), In some embodiments, the methods described herein prevent, delay, or attenuate the development of fibrosis in a subject at risk for developing fibrosis (e.g., a subject undergoing chemotherapy, radiation, or surgery, or a subject suffering from 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 involve affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in a subject suffering from fibrosis or a disease or condition associated with fibrosis.

[0230] 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 (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, bluish coloration of the lips or skin (cyanosis), dizziness, or syncope), prevent PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) (e.g., prevent its development). In some embodiments, a polypeptide described herein can be administered to slow or stop PH (e.g., PAH, venous PH, hypoxic PH, The polypeptides described herein can reduce the symptoms of PH (e.g., shortness of breath (dyspnea), fatigue, swelling (e.g., edema) of the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, bluish color of the lips or skin (cyanosis), dizziness, or syncope), or slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), compared to the symptoms or progression observed before treatment, or compared to the symptoms or progression of PH in an untreated subject.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 disease or condition that results in an increased risk of developing PAH), such as HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary angiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use); the subject may have a family history of PH (e.g., heritable PAH); the subject may have a disease or condition associated with venous PH (e.g., a disease or condition that results in an increased 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 disease or condition associated with hypoxic PH (e.g., a disease or condition that results in an increased risk of developing hypoxic PH), such as chronic obstructive pulmonary disease (COPD); 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 artery stenosis, or parasitic infection); Or the subject may have a disease or condition associated with other PH (e.g., a disease or condition that results in an increased risk of developing other PH), such as a hematologic 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), thrombotic microangiopathy of the lung, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).In some embodiments, the polypeptides described herein prevent, delay, or attenuate the development of PH in a subject at risk of developing PH (e.g., a subject with a family history of PH (e.g., heritable PAH), or a subject with a condition that predisposes to the development of PAH (e.g., HIV infection, schistosomiasis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use), venous PH (e.g., 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), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, chronic exposure to high altitude or developmental abnormalities), thromboembolic PH (e.g., chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital In some embodiments, the methods described herein relate to affecting the development of myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in subjects with 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).

[0231] In some embodiments, polypeptides comprising an extracellular ActRIIA variant described herein reduce or inhibit the binding of myostatin, activin, and / or BMP9 to their endogenous receptors, such as ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIA). The polypeptides described herein can reduce the binding of myostatin, activin, and / or BMP9 to their endogenous receptors as compared to the binding of myostatin, activin, and / or BMP9 to their endogenous receptors in the absence of the polypeptides of the invention. In some embodiments, affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors, e.g., ActRIIA, ActRIIB and BMPRII (e.g., ActRIIA)) results in an increase in red blood cell levels (e.g., hemoglobin levels, hematocrit levels, or red blood cell counts, e.g., inducing or increasing red blood cell formation and / or red blood cell mass) in a subject, a reduction in fibrosis or risk of developing fibrosis in a subject, a delay in the development of fibrosis, a slowing or halting of the progression of fibrosis, a reversal of fibrosis, a reduction in symptoms of PH in a subject (e.g., a reduction in symptoms such as shortness of breath (dyspnea), fatigue, swelling (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, a rapid pulse or palpitations, a bluish color to the lips or skin (cyanosis), dizziness or syncope), a reduction in the risk of developing PH, a delay in the development of PH, and / or a slowing or halting of the progression of PH in a subject. PH can be PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.

[0232] In some embodiments, the polypeptides described herein (e.g., including extracellular ActRIIA variants (e.g., having SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: A polypeptide of an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 6-72), e.g., an effective amount of an ActRIIA variant, can be administered to a subject to increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, increase red blood cell count, increase red blood cell mass, or induce or increase erythropoiesis), prevent or reduce fibrosis (e.g., reduce fibrosis, prevent, delay or attenuate the development of fibrosis, slow or stop the progression of fibrosis, or reverse fibrosis), prevent or treat PH (e.g., reduce symptoms of PH, prevent, delay or attenuate the development of PH, or slow or stop the progression of PH, such as PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), or affect myostatin, activin, and / or BMP9 signaling in a subject. An extracellular ActRIIA variant (e.g., 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 to increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, increase red blood cell count, increase red blood cell mass, or induce or increase erythropoiesis), prevent or reduce fibrosis (e.g., reduce fibrosis, prevent, delay or attenuate the development of fibrosis, slow or stop the progression of fibrosis, or reverse fibrosis), prevent or treat PH (e.g., reduce symptoms of PH, prevent, delay or attenuate the development of PH, or slow or stop the progression of PH, such as PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), or affect myostatin, activin, and / or BMP9 signaling in a subject. An extracellular ActRIIA variant (e NO: 6-72), such as an effective amount of an ActRIIA variant) can increase red blood cell levels, prevent or reduce fibrosis, or prevent or treat PH. In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage. In some embodiments of the methods described herein, the subject has a disease or condition or is at risk of developing a disease or condition that involves low red blood cell levels (e.g., anemia or blood loss, such as anemia associated with cancer (e.g., multiple myeloma, leukemia, breast cancer, lung cancer, colon cancer), cancer treatment (e.g., chemotherapy or radiotherapy), 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 such as 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 involving fibrosis (e.g., chemotherapeutic drug-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, osteoarticular fibrosis, 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, schistosomiasis infection, kidney disease, heart disease, macular degeneration, retino- or vitreoretinopathy, systemic or localized scleroderma, atherosclerosis, or restenosis). In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition involving PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH, such as idiopathic PAH; heritable PAH; PAH associated with or caused by HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use); venous PH associated with or caused by left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; hypoxic PH 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, prolonged exposure to high altitude, or developmental abnormalities; thromboembolic PH associated with or caused by 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 caused by hematologic disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic disorders (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), thrombotic microangiopathy of the lung, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).

[0233] The present invention also includes methods of treating a subject having anemia or blood loss 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 having low red blood cell levels (e.g., low hemoglobin levels or low red blood cell count, e.g., low red blood cell mass) or 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) has anemia or blood loss or is at risk of developing anemia or blood loss. In some In some embodiments, the anemia is associated with or caused by nutritional deficiencies (e.g., vitamin deficiencies), bone marrow defects (e.g., paroxysmal nocturnal hemoglobinuria), adverse reactions to drug therapy (e.g., antiretroviral HIV drugs), myelodysplastic syndrome, bone marrow transplantation, cancer (e.g., solid tumors such as breast cancer, lung cancer, colon cancer; lymphatic system tumors such as chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma; or hematopoietic system tumors such as leukemia or multiple myeloma), cancer treatment (e.g., radiation or chemotherapy, e.g., such as chemotherapy with platinum-containing agents), inflammatory or autoimmune diseases (e.g., rheumatoid arthritis, other inflammatory arthritis, systemic lupus erythematosus (SLE), acute or chronic skin diseases (e.g., psoriasis), or inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), cystitis, gastritis), acute or chronic kidney disease or failure (e.g., chronic kidney disease), including idiopathic or congenital conditions, acute or chronic liver disease, diabetes, acute or chronic bleeding, infection (e.g., malaria, osteomyelitis), splenomegaly, porphyria, vasculitis, hemolysis, urinary tract infection, hemoglobin In some embodiments, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with a bone marrow disorder, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, DeBouchet-Jacques anemia, Fanconi anemia, or refractory anemia with blasts.Compositions and methods as described herein can also be used to treat subjects who do not respond well to erythropoietin (EPO), or are susceptible to the adverse effects of EPO (such as hypertension, headache, vascular thrombosis, flu-like syndrome, shunt obstruction and myocardial infarction). In some embodiments, blood loss is due to surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations or excessive menstrual bleeding (such as menorrhagia). In some embodiments, compared with the measured values obtained before treatment, the methods described herein increase red blood cell levels (such as hemoglobin levels, hematocrit or red blood cell count, such as red blood cell volume). In some embodiments, compared with the measured values obtained before treatment, the methods described herein increase or induce red blood cell formation. In some embodiments, compositions and methods as described herein reduce the needs of subjects about blood transfusion (such as subjects no longer need blood transfusion, or compared with before treatment with compositions and methods as described herein, subjects need less frequent blood transfusion). Subjects with normal red blood cell levels can also be treated using the methods and compositions described herein to increase red blood cell levels so that blood can be extracted and stored for use in transfusions later.

[0234] The present invention also includes methods of 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 or is at risk of developing fibrosis. In some embodiments, the fibrosis is chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis (e.g., cystic fibrosis, idiopathic fibrosis, or fibrosis associated with tuberculosis, pneumonia, or coal dust), liver fibrosis (e.g., cirrhosis, biliary atresia), renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis (e.g., endomyocardial fibrosis), In some embodiments, the fibrosis is associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitrectomy. In some embodiments, the subject is at risk for developing fibrosis associated with cancer treatment (chemotherapy or radiation), disease or infection (e.g., tuberculosis, pneumonia, myocardial infarction, hepatitis B or C infection, fatty liver disease, schistosomiasis 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, wounds, or burns. In some embodiments, the fibrosis in the subject is measured compared to measurements obtained before treatment or compared to measurements in untreated subjects. In some embodiments, the methods described herein reduce fibrosis compared to treatment with a subject who has undergone treatment with a fibrotic disorder. In some embodiments, the methods described herein prevent the development of fibrosis, reduce the risk of developing fibrosis (e.g., reducing the risk of developing fibrosis compared to the development of fibrosis in an untreated subject), or reverse existing fibrosis. In some embodiments, the methods described herein slow, stop, or reverse the progression of fibrosis (e.g., slowing the progression of fibrosis compared to the progression before treatment, or compared to the progression in an untreated or untreated subject). In some embodiments, the methods described herein improve organ or tissue function (e.g., function of an organ or tissue with fibrosis) compared to organ or tissue function before treatment.Tissue and organ function can be assessed using any of the standard clinical tests commonly used to evaluate tissue and organ function.

[0235] The present invention also includes methods of treating a subject having PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) or at risk of developing 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 may have PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) or at risk of developing PH. In some embodiments, the PH is PAH. In some embodiments, the PAH is idiopathic PAH. In some embodiments, the PAH is heritable PAH. In some embodiments, the PAH is idiopathic PAH. AH is PAH associated with (e.g., caused by or associated with) HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use). In some embodiments, PH is venous PH. In some embodiments, venous PH is venous PH associated with (e.g., caused by or associated with) left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired Pulmonary vein stenosis. In some embodiments, PH is hypoxic PH. In some embodiments, hypoxic PH is hypoxic PH associated with (e.g., caused by or associated with) 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, long-term exposure to high altitude, or developmental abnormalities. In some embodiments, PH is thromboembolic PH. In some embodiments, thromboembolic PH is thromboembolic PH associated with (e.g., caused by or associated with) chronic thromboembolic pulmonary hypertension, or other pulmonary Arterial obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection). In some embodiments, PH is other PH. In some embodiments, other PH is other PH associated with (e.g., caused by or associated with) a hematologic 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 tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).In some embodiments, the methods described herein reduce symptoms of PH (e.g., reduce the severity or frequency of symptoms such as shortness of breath (dyspnea), fatigue, swelling (e.g., edema) of the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, bluish color to the lips or skin (cyanosis), dizziness, or syncope, compared to the frequency or severity of symptoms before treatment). In some embodiments, the methods described herein prevent the development 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 development of PH in an untreated subject). In some embodiments, the methods described herein slow or stop 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 of a subject (e.g., the initiation or progression of vascular remodeling in the heart or lungs) 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 the progression of right ventricular hypertrophy) compared to right ventricular hypertrophy before treatment or compared to right ventricular hypertrophy in an untreated subject. Symptoms of PH can be assessed using standard clinical tests before and after treatment. Common tests for assessing PH include electrocardiograms, pulmonary function tests, echocardiograms, right heart catheterizations, computed tomography scans, measurements of pulmonary vascular resistance, and a 6-minute walk test. In some embodiments, the methods described herein reduce pulmonary vascular resistance (e.g., resulting in a reduction in pulmonary vascular resistance compared to pulmonary vascular resistance before treatment). 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.

[0236] 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)) further comprising a C-terminal extension of one to six amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids) can be used as a therapeutic protein. In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) 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 a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) can be used as a therapeutic protein. Nucleic acids encoding the polypeptides described herein or vectors containing the nucleic acids can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptides, nucleic acids, or vectors can be administered as part of a pharmaceutical composition. Compositions that can be administered to a subject according to the methods described herein are provided in Table 4 below.

[0237] Table 4

[0238]

[0239]

[0240]

[0241] Example

[0242] Example 1 - Assessment of ActRIIA variant binding affinity by surface plasmon resonance (SPR)

[0243] Biacore 3000 was used to measure the kinetics of the interaction between ActRIIA variants and the ligands activin A, activin B, growth differentiation factor 11 (GDF11), and BMP-9. ActRIIA variants were prepared by transient expression in HEK293 cells and purified from conditioned medium using protein-A Sepharose chromatography. ActRIIA variants were immobilized on a chip (CM4 or CM5) in flow chambers 2-4 with capture antibodies (anti-mouse from GEGE) to ensure correct orientation. Flow chamber 1 was used as a reference chamber to subtract any nonspecific binding and volume effects. HBS-EP + buffer from GE Healthcare™ was used as running buffer. Each ligand was run in a concentration series at 40 μl / min to avoid mass transfer effects. Data were analyzed using Scrubber2 of BioLogic™ software to calculate the K for each interaction. D (Table 5).

[0244] Table 5: Affinity of ActRIIA variants to various ligands (K D )

[0245]

[0246] Example 2 - Effect of ActRIIA / B-Fc on fibrosis in mdx mice

[0247] The effect of ActRIIA / B-Fc (SEQ ID NO: 69 fused to an Fc domain) on reducing intramuscular fibrosis associated with muscular dystrophy was analyzed using mdx mice, a murine model of Duchenne muscular dystrophy. Briefly, female C57Bl / 10 and mdx mice aged 12-13 months were treated twice weekly with vehicle or 20 mg / kg ActRIIA / B-Fc for 12 weeks. During necropsy, the diaphragm and quadriceps muscles were collected and immediately snap-frozen in liquid nitrogen. Protein was then extracted from the tissue and hydrolyzed, followed by high performance liquid chromatography in a Hitachi L8900 amino acid analyzer. Individual amino acid content was determined by comparing chromatographic peaks against known standards. Hydroxyproline, an amino acid unique to collagen and a surrogate for total fibrotic content, was calculated as mg / gram of total protein extracted. Figure 2 As shown in . Treatment with ActRIIA / B-Fc attenuates the development of fibrosis in aged mdx mice.

[0248] Example 3 - Effect of ActRIIA / B-Fc on Red Blood Cell Mass in Nonhuman Primates

[0249] Two to three year old cynomolgus monkeys were treated with vehicle or 3, 10, or 50 mg / kg ActRIIA / B-Fc (SEQ ID NO: 69 fused to the Fc domain) every two weeks for three months. On day 92, blood was collected in K2EDTA tubes and assayed on an Advia hematology analyzer. Figure 3 As shown in Figure 2, 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.

[0250] Example 4 - Effects of Extracellular ActRIIA Variants on PAH

[0251] In one experiment, PAH was induced in male rats using a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg). To determine whether treatment with an ActRIIA variant could prevent the development of PAH, rats were randomly assigned to a vehicle or ActRIIA variant treatment group 24 hours after PAH induction and treated with an ActRIIA variant (5 or 15 mg / kg) or vehicle twice a week for 21 days. On day 14, ventricular function and right ventricular (RV) remodeling were examined by electrocardiography, which was performed by anesthetizing the rats with 1.5% isoflurane and using a small animal high-frequency ultrasound probe to detect pulmonary blood flow acceleration, right ventricular function and hypertrophy, and left ventricular function while the animals were kept in a supine position. Doppler across the mitral and tricuspid valves was used to determine whether treatment with an ActRIIA variant induced any significant regurgitation or lesions. On day 21, the rats were anesthetized with pentobarbital, intubated through the trachea, and mechanically ventilated using a rodent ventilator. Hemodynamics were assessed using a fluid-filled catheter through the RV apex. Rats were perfused with PBS followed by 1% paraformaldehyde (PFA). To measure RV hypertrophy (RVH), the heart was excised and the RV wall was removed from the left ventricle plus septum (LV+S) and weighed separately. The extent of RVH was determined by the ratio RV / (LV+S).

[0252] In a second experiment, PAH was induced in male rats using a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg). To determine whether treatment with an ActRIIA variant could slow or reduce the progression of PAH, rats were injected again with MCT on day 18 and randomly assigned to vehicle or ActRIIA variant treatment groups. Rats were injected with an ActRIIA variant (15 mg / kg) or vehicle three times per week. Hemodynamics and RVH were examined on day 35 as described above.

[0253] Example 5 - Treatment of Anemia by Administration of Extracellular ActRIIA Variants

[0254] 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 that measures hematological parameters. To treat a 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 a sequence of any one 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 anemic conditions. 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)) is administered in a therapeutically effective amount, such as 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered once every two months, 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 times a week or more). The extracellular ActRIIA variant is administered in an amount sufficient to increase hemoglobin levels, increase red blood cell count, or increase hematocrit.

[0255] After the composition is administered to a patient, those skilled in the art can monitor the patient's improvement in response to therapy by various methods. For example, a doctor can monitor the patient's hemoglobin level, red blood cell count, or hematocrit by performing a blood test. A finding that the patient shows improved hemoglobin level, red blood cell count, or hematocrit after the composition is administered, compared to the test results before the composition is administered, indicates that the patient is responding well to the treatment. Subsequent dosages can be determined and administered as needed.

[0256] Example 6 - Treatment of fibrosis by administration of extracellular ActRIIA variants

[0257] 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 stop 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 to the subject a composition containing an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). 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) to fibrotic tissue or organ. 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)) is administered in a therapeutically effective amount, such as 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered once every two months, 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 times a week or more). The extracellular ActRIIA variant is administered in an amount sufficient to reduce the symptoms of fibrosis or slow or stop the progression of fibrosis.

[0258] After the composition is administered to the patient, those skilled in the art can monitor the improvement of the patient's response to therapy by various methods. For example, a doctor can monitor the patient's fibrosis by performing an imaging test, and can use standard clinical tests to monitor the patient's symptoms. Compared to the test results before the composition is administered, the finding that the patient's symptoms are reduced or the progression of the patient's fibrosis is slowed or stopped after the composition is administered indicates that the patient is responding well to the treatment. Subsequent doses can be determined and administered as needed.

[0259] Example 7 - Treatment of pulmonary hypertension by administration of extracellular ActRIIA variants

[0260] 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. Treatment methods 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, or 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 the sequence of any one 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 the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) is administered in a therapeutically effective amount, such as 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered once every two months, 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 times a week or more). The extracellular ActRIIA variant is administered in an amount sufficient to reduce the symptoms of PH or slow or stop the progression of PH.

[0261] After the composition is administered to the patient, those skilled in the art can monitor the patient's improvement in response to therapy by various methods. For example, a doctor can use standard clinical tests and patient self-reports to monitor the patient's symptoms. A finding that the patient's PH symptoms are reduced or the patient's PH progression is slowed or stopped after administration of the composition, compared to the test results before administration of the composition, indicates that the patient is responding well to treatment. Subsequent doses can be determined and administered as needed.

[0262] Other implementation plans

[0263] While the invention has been described with reference to specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention which follow from the principles of the invention in general and include such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and which may be applied to the basic features herein above described.

[0264] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0265] Other embodiments are within the following claims.

Claims

1. Use of a polypeptide consisting of the sequence of GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCW LDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 69) and a human IgG1 Fc domain monomer linked to the C-terminus of SEQ ID NO: 69 in the preparation of a medicament for treating anemia in a human subject, wherein the anemia is anemia associated with myelodysplastic syndrome or anemia associated with myelofibrosis.

2. Use of a polypeptide consisting of the sequence of GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCW LDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 69), a human IgG1 Fc domain monomer, and a linker polypeptide connecting the Fc domain monomer to the C-terminus of SEQ ID NO: 69 in the preparation of a medicament for treating anemia in a human subject, wherein the anemia is anemia associated with myelodysplastic syndrome or anemia associated with myelofibrosis.

3. Use of a polypeptide according to claim 1 or 2, wherein the polypeptide is in the form of a homodimer.

4. Use of the polypeptide according to claim 2, wherein the linker polypeptide has 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 IDNO: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), 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), GGSGGGSGGGSG (SEQ IDNO: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 IDNO:137), AEAAAKEAAAKA(SEQ ID NO:138), KESGSVSSEQLAQFRSLD(SEQ ID NO:139), GENLYFQSGG(SEQ ID NO:140), SACYCELS(SEQ ID NO:141), RSIAT(SEQID). NO:142)、RPACKIPNDLKQKVMNH(SEQ ID NO:143)、GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG(SEQ ID NO:144)、AAANSSIDLISVPVDSR(SEQ ID NO:145)、oggsgggsggggedgggggsggggggggggggggggggggsggggggggggggggggsg (seq id NO:146) with EAAAK(SEQ ID NO:147) and PAPAP(SEQ ID NO:148) gene.

5. Use of a polypeptide according to claim 4, wherein the linker polypeptide has an amino acid sequence of GGG.

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