ACTRII protein for the treatment of pulmonary arterial hypertension (PAH)

Abstract

In some aspects, the present disclosure relates to compositions and methods comprising an ActRII polypeptide for treating, preventing, or reducing the rate of progression and / or severity of pulmonary arterial hypertension, particularly for treating, preventing, or reducing the rate of progression and / or severity of one or more pulmonary arterial hypertension-related complications. The polypeptide may be a lyophilized polypeptide and may be contained in a vial to form a kit.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the benefit of priority based on U.S. Provisional Application No. 63 / 042,722, filed on June 23, 2020; U.S. Provisional Application No. 63 / 084,409, filed on September 28, 2020; U.S. Provisional Application No. 63 / 112,513, filed on November 11, 2020; and U.S. Provisional Application No. 63 / 188,141, filed on May 13, 2021. The disclosures of the foregoing applications are hereby incorporated herein by reference in their entireties.

Background Art

[0002] Background of the Invention Pulmonary hypertension (PH) is a disease characterized by high blood pressure in the pulmonary vascular system, including the pulmonary arteries, pulmonary veins, and pulmonary capillaries. Generally, PH is defined as a mean pulmonary artery pressure (mPAP) of ≥20 mmHg at rest or ≥30 mmHg during exercise [Hill et al., Respiratory Care 54(7):958 - 68 (2009)]. One of the main symptoms of PH is dyspnea or shortness of breath, and other symptoms include fatigue, dizziness, syncope, peripheral edema (swelling of the feet, lower legs or ankles), bluish lips and skin, chest pain, angina, mental confusion during exercise, dry cough, increased pulse, and palpitations. PH can be a severe disease that causes heart failure, which is one of the most common causes of death in people with pulmonary hypertension. Post - operative pulmonary hypertension can make many types of surgeries or procedures difficult and presents problems associated with high mortality.

[0003] Public health (PH) can be grouped based on the different manifestations of diseases that share similarities in pathophysiological mechanisms, clinical presentation, and therapeutic approaches [Simonneau et al., JACC 54(1):S44-54 (2009)]. The clinical classification of PH was first proposed in 1973, and the most recently updated clinical classification was approved by the World Health Organization (WHO) in 2018. According to the latest updated clinical classification of PH, there are five main groups of PH: pulmonary arterial hypertension (PAH) characterized by pulmonary artery wedge pressure (PAWP) of ≤15 mmHg; PH resulting from left heart disease (also known as pulmonary venous hypertension or congestive heart failure) characterized by PAWP >15 mmHg; PH resulting from lung disease and / or hypoxia; PH resulting from pulmonary artery occlusion; and PH due to unclear and / or multifactorial etiologies [Simonneau et al., JACC 54(1):S44-54 (2009); Hill et al., Respiratory Care 54(7):958-68 (2009)]. PAH is further classified into idiopathic PAH (IPAH), which is a sporadic disorder with no family history of PAH or identified risk factors; hereditary PAH; drug and toxin-induced PAH; PAH associated with connective tissue disorders, HIV infection, portal hypertension, congenital heart defects, schistosomiasis, and chronic hemolytic anemia; and persistent PH in neonatal patients [Simonneau et al., (2019) Eur Respir J: 53:1801913]. Diagnosis of the various types of PH requires a series of tests.

[0004] Generally, the treatment of pulmonary hypertension (PH) depends on the cause or classification of the PH. When PH is caused by a known drug or medical condition, this is known as secondary PH, and its treatment is usually directed towards the underlying disease. Treatment of group 2 pulmonary hypertension (e.g., venous hypertension) generally involves optimizing left ventricular function by administering diuretics, beta-blockers, and ACE inhibitors, or by repairing or replacing the mitral or aortic valve. PAH therapy includes pulmonary vasodilators, digoxin, diuretics, anticoagulants, and oxygen therapy. Pulmonary vasodilators target different pathways, including the prostacyclin pathway (e.g., prostacyclins including intravenous epoprostenol, subcutaneous or intravenous treprostinil, and inhaled iloprost), the nitric oxide pathway (e.g., phosphodiesterase-5 inhibitors including sildenafil and tadalafil), and the endothelin-1 pathway (e.g., endothelin receptor antagonists including oral bosentan and oral ambrisentan) [Humbert, M. Am. J. Respir. Crit. Care Med. 179:650-6 (2009); Hill et al., Respiratory Care 54(7):958-68 (2009)]. However, current therapies do not result in a cure for PH and do not directly address the underlying vascular remodeling and vascular muscularization observed in many PH patients.

[0005] There is a high and unmet need for effective treatments for pulmonary hypertension. Therefore, the object of this disclosure is to provide methods for treating, preventing, or reducing the rate of progression and / or severity of PH, in particular, methods for treating, preventing, or reducing the rate of progression and / or severity of one or more PH-related complications. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Hill et al., Respiratory Care 54(7):958-68 (2009) [Non-Patent Document 2] Simonneau et al., JACC 54(1):S44-54 (2009) [Non-Patent Document 3] Simonneau et al., (2019) Eur Respir J: 53:1801913 [Non-Patent Document 4] Humbert, M. Am. J. Respir. Crit. Care Med. 179:650-6 (2009) [Overview of the project] [Means for solving the problem]

[0007] Summary of the Invention In certain embodiments, the Disclosure relates to a method for treating pulmonary arterial hypertension (PAH), comprising the step of administering a therapeutically effective dose of ActRII polypeptide to a patient, wherein the polypeptide has an amino acid sequence beginning with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1 and ending with any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 of SEQ ID NO: 1, and at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% The present invention provides a method for administering a polypeptide comprising an amino acid sequence that is 97%, 98%, 99%, or 100% identical, in a dosage range of 0.1 mg / kg to 2.0 mg / kg, wherein the administration of the polypeptide results in a change in one or more hemodynamic or functional parameters, including: a reduction in pulmonary vascular resistance (PVR); an increase in the 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the functional class of pulmonary hypertension as recognized by the World Health Organization (WHO); promotion or increase of regression of the functional class of pulmonary hypertension as recognized by the WHO; improvement in right ventricular function; improvement in pulmonary artery pressure; and / or improvement in mean right atrial pressure.

[0008] In certain embodiments, the Disclosure provides a method for treating, preventing, or reducing the rate and / or severity of progression of one or more complications of pulmonary arterial hypertension, comprising the step of administering an effective amount of ActRII polypeptide to a patient in need thereof, wherein the polypeptide has an amino acid sequence beginning with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1 and ending with any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 of SEQ ID NO: 1, and contains at least 75%, 80%, 85%, or 90% The present invention provides a method for administering a polypeptide comprising an amino acid sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, in a dosage range of 0.1 mg / kg to 2.0 mg / kg, wherein the administration of the polypeptide results in a change in one or more hemodynamic or functional parameters, including: a reduction in pulmonary vascular resistance (PVR); an increase in the 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the functional class of pulmonary hypertension as recognized by the World Health Organization (WHO); promotion or increase of regression of the functional class of pulmonary hypertension as recognized by the WHO; improvement in right ventricular function; improvement in pulmonary artery pressure; and / or improvement in mean right atrial pressure. In some embodiments, one or more complications of pulmonary arterial hypertension are selected from the group consisting of smooth muscle and / or endothelial cell proliferation in the pulmonary arteries, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular remodeling, right ventricular hypertrophy, and pulmonary fibrosis.

[0009] In certain embodiments, the Disclosure relates to a method for treating pulmonary arterial hypertension (PAH), comprising the step of administering a therapeutically effective dose of an ActRII polypeptide regimen to a patient, wherein the polypeptide begins with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1, and continues through amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, The present invention provides a method comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence ending at any one of 130, 131, 132, 133, 134, or 135, wherein the regimen comprises a first dose of polypeptide between 0.1 mg / kg and 1.0 mg / kg over a first period, and a second dose of polypeptide between 0.1 mg / kg and 1.0 mg / kg thereafter administered over a second period. In some embodiments, polypeptide administration results in a change in one or more hemodynamic or functional parameters, including: a reduction in pulmonary vascular resistance (PVR); an increase in the 6-minute walk distance (6MWD); a decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or reduction of progression of the functional class of pulmonary hypertension as recognized by the World Health Organization (WHO); promotion or increase of regression of the functional class of pulmonary hypertension as recognized by the WHO; improvement in right ventricular function; improvement in pulmonary artery pressure; and improvement in mean right atrial pressure. In some embodiments, the first period is at least 3 weeks. In some embodiments, the second period is at least 3 weeks. In some embodiments, the second period is at least 21 weeks. In some embodiments, the second period is at least 45 weeks. In some embodiments, the second period exceeds the first period. In some embodiments, the second dose exceeds the first dose. In some embodiments, the first dose is in the range of about 0.2 mg / kg to about 0.4 mg / kg, followed by a second dose in the range of about 0.5 mg / kg to about 0.8 mg / kg.In some embodiments, the first dose is approximately 0.3 mg / kg, followed by a second dose of approximately 0.7 mg / kg.

[0010] In some embodiments, the method reduces PVR in a patient. In some embodiments, the method reduces PVR in a patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the method reduces PVR in a patient by at least 20%. In some embodiments, the reduction in PVR is a result of reduced mean pulmonary artery pressure. In some embodiments, the method increases the patient's 6-minute walk distance. In some embodiments, the method increases the patient's 6-minute walk distance by at least 10 meters (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300 meters, or more than 400 meters). In some embodiments, the method increases the patient's 6-minute walk distance by at least 30 meters. In some embodiments, the method reduces NT-proBNP levels in a patient. In some embodiments, the method reduces NT-proBNP levels in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, the method reduces NT-proBNP levels in the patient by at least 30%. In some embodiments, the method reduces NT-proBNP levels to normal levels. In some embodiments, the normal level of NT-proBNP is <100 pg / ml.

[0011] In some embodiments, the method prevents or reduces the progression of the functional class of pulmonary hypertension as recognized by the WHO. In some embodiments, the method prevents or reduces the progression of the functional class of pulmonary hypertension from functional class I to functional class II as recognized by the WHO. In some embodiments, the method prevents or reduces the progression of the functional class of pulmonary hypertension from functional class II to functional class III as recognized by the WHO. In some embodiments, the method prevents or reduces the progression of the functional class of pulmonary hypertension from functional class III to functional class IV as recognized by the WHO. In some embodiments, the method promotes or increases the regression of the functional class of pulmonary hypertension as recognized by the WHO. In some embodiments, the method promotes or increases the regression of the functional class of pulmonary hypertension from functional class IV to functional class III as recognized by the WHO. In some embodiments, the method promotes or increases the regression of the functional class of pulmonary hypertension from functional class III to functional class II as recognized by the WHO. In some embodiments, the method promotes or increases the regression of the functional class of pulmonary hypertension from functional class II to functional class I as recognized by the WHO.

[0012] In some embodiments, the method improves right ventricular function in the patient. In some embodiments, the improvement in right ventricular function is due to an increase in the rate of change of right ventricular area. In some embodiments, the improvement in right ventricular function is due to a decrease in right ventricular hypertrophy. In some embodiments, the improvement in right ventricular function is due to an increase in the ejection fraction. In some embodiments, the improvement in right ventricular function is due to an increase in both the rate of change of right ventricular area and the ejection fraction.

[0013] In some embodiments, the method improves pulmonary artery pressure in a patient. In some embodiments, the improvement in pulmonary artery pressure is a reduction in mean pulmonary artery pressure (mPAP). In some embodiments, the method reduces mPAP in a patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the method reduces mPAP in a patient by at least 3 mmHg (e.g., at least 3%, 5%, 7%, 10%, 12%, 15%, 20%, or 25 mmHg). In some embodiments, the method improves mean right atrial pressure (mRAP) in a patient. In some embodiments, the improvement in mRAP is a reduction in mRAP. In some embodiments, the method reduces mRAP in a patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the method reduces mRAP in the patient by at least 1 mmHg (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mmHg).

[0014] In some embodiments, the patient has a pulmonary vascular resistance (PVR) greater than or equal to 3 Wood units. In some embodiments, the patient has a 6-minute walking distance of 150 to 550 meters. In some embodiments, the patient has elevated NT-proBNP levels compared to healthy patients. In some embodiments, the patient has an NT-proBNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the patient has elevated brain natriuretic peptide (BNP) levels compared to healthy patients. In some embodiments, the patient has a BNP level of at least 100 pg / mL (e.g., 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the method reduces the BNP level in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, the method reduces the BNP level to a normal level (i.e., <100 pg / mL). In some embodiments, the patient has an mPAP selected from the group consisting of mean pulmonary artery pressure (mPAP) of at least 20 mmHg; mPAP of at least 25 mmHg; mPAP of at least 30 mmHg; mPAP of at least 35 mmHg; mPAP of at least 40 mmHg; mPAP of at least 45 mmHg; and mPAP of at least 50 mmHg. In some embodiments, the patient has an mRAP selected from the group consisting of mean right atrial pressure (mRAP) of at least 5 mmHg; mRAP of at least 6 mmHg; mRAP of at least 8 mmHg; mRAP of at least 10 mmHg; mRAP of at least 12 mmHg; mRAP of at least 14 mmHg; and mRAP of at least 16 mmHg.

[0015] In some embodiments, PAH is idiopathic pulmonary arterial hypertension (PAH). In some embodiments, PAH is hereditary PAH. In some embodiments, PAH is drug or toxin-induced PAH. In some embodiments, PAH is PAH associated with a simple congenital systemic-to-pulmonary shunt at least one year after shunt repair. In some embodiments, the patient has pulmonary hypertension of function class II or class III according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has pulmonary hypertension of function class I, class II, class III or class IV as recognized by the World Health Organization. In some embodiments, the patient has pulmonary hypertension of function class I, class II, class III or class IV according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the patient has pulmonary hypertension of function class IV according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the method increases transplant-free survival in the patient. In some embodiments, the method increases transplant-free survival in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the method reduces right ventricular hypertrophy in patients. In some embodiments, the method reduces right ventricular hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the method reduces smooth muscle hypertrophy in patients. In some embodiments, the method reduces smooth muscle hypertrophy in patients by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the method reduces pulmonary arteriole muscularization in patients. In some embodiments, the method reduces pulmonary arteriole muscularization in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0016] In some embodiments, the method increases the patient's exercise capacity. In some embodiments, the method reduces the patient's Borg dyspnea index (BDI). In some embodiments, the method reduces the patient's BDI by at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points). In some embodiments, the patient has reduced renal function. In some embodiments, the method further improves renal function. In some embodiments, the method delays the clinical progression of pulmonary arterial hypertension. In some embodiments, the method delays the clinical progression of pulmonary arterial hypertension according to the World Health Organization's functional classification system for pulmonary hypertension. In some embodiments, the method reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension. In some embodiments, the method reduces the risk of pathological conditions for one or more complications associated with pulmonary arterial hypertension. In some embodiments, the method includes changes in the pathological condition, such as an increased need for lung and / or heart transplantation; the need to initiate rescue therapy with known procedures for PAH; the need to increase prostacyclin by at least 10%; the need for atrial septal dehiscence; PAH-specific hospitalization for at least 24 hours; and an exacerbation of PAH. In some embodiments, the exacerbation of PAH includes a deterioration in the class of WHO function and a decrease of at least 15% in 6MWD. In some embodiments, the method reduces the risk of death associated with pulmonary arterial hypertension. In some embodiments, the method reduces the risk of death associated with pulmonary arterial hypertension by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, the patient has hemoglobin levels >8 and <15 g / dl. In some embodiments, the patient's hemoglobin level is <18 g / dl.

[0017] In some embodiments, the ActRII polypeptide includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the ActRII polypeptide includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the ActRII polypeptide is a fusion protein further comprising the Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In some embodiments, the Fc fusion protein further comprises a linker domain located between the ActRII polypeptide domain and the Fc domain of the immunoglobulin. In some embodiments, the linker domain is selected from the group consisting of TGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO: 21). In some embodiments, the ActRII polypeptide contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.In some embodiments, the polypeptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the polypeptide is lyophilized. In some embodiments, the polypeptide is soluble. In some embodiments, the polypeptide is administered to the patient by subcutaneous injection. In some embodiments, the polypeptide is administered to the patient every three weeks. In some embodiments, the polypeptide is administered to the patient every four weeks. In some embodiments, the ActRII polypeptide is administered to the patient weekly, every two weeks, every three weeks, or every four weeks. In some embodiments, the ActRII polypeptide is administered to the patient every three weeks. In some embodiments, the polypeptide is part of a homodimeric protein complex. In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide has a glycosylation pattern that can be obtained by expression in Chinese hamster ovary cells. In some embodiments, the ActRII polypeptide binds to one or more ligands selected from the group consisting of activin A, activin B, and GDF11. In some embodiments, the ActRII polypeptide binds to activin and / or GDF11. In some embodiments, the ActRII polypeptide further binds to one or more ligands selected from the group consisting of BMP10, GDF8, and BMP6.

[0018] In some embodiments, the ActRII polypeptide is administered in a dose between 0.1 mg / kg and 2.0 mg / kg. In some embodiments, the ActRII polypeptide is administered in a dose of 0.3 mg / kg. In some embodiments, the ActRII polypeptide is administered in a dose of 0.7 mg / kg. In some embodiments, the method includes the step of further administering additional activators and / or supportive therapies to the patient. In some embodiments, the additional activators and / or supportive therapies are selected from the group consisting of beta-blockers, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), diuretics, lipid-lowering agents, endothelin blockers, PDE5 inhibitors, prostacyclins, or left ventricular assist devices (LVADs). In some embodiments, additional activators and / or supportive therapies include: prostacyclins and their derivatives (e.g., epoprostenol, treprostinil, and iloprost); prostacyclin receptor agonists (e.g., selexipag); endothelin receptor antagonists (e.g., therin, ambrisentan, macitentan, and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem, and nifedipine); anticoagulants (e.g., warfarin); diuretics; oxygen therapy; atrial septal dehiscence; pulmonary endarterectomy; phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil); soluble guanylate cyclase activators (e.g., synaciguat and riociguat); ASK -1 inhibitors (e.g., CIIA; SCH79797; GS-4997; MSC2032964A; 3H-naphtho[1,2,3-de]quinilin-2,7-dione, NQDI-1; 2-thioxo-thiazolidine, 5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indole-2-one); NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2,2-difluoropropionamide; C28-imidazole (CDDO-Im); 2-cyano-3,12-dioxolean-1,9-diene-28-euic acid (CDDO); 3-acetyloleanolic acid; 3-trifluoroacetyloleanolic acid);28-methyl-3-acetyloleanane; 28-methyl-3-trifluoroacetyloleanane; 28-methyloxyoleanolic acid; SZC014; SCZ015; SZC017; PEGylated derivative of oleanolic acid; 3-O-(beta-D-glucopyranosyl)oleanolic acid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid; 3 -O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester; 3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta- D-Glucopyranosyl ester; 3-O-[aL-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid; 3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester; 28-O-β-D-glucopyranosyl-oleanolic acid; 3-O-β-D-glucopyranosyl Nosyl(1→3)-β-D-glucopyranosidouronic acid (CS1); oleanolic acid 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosidouronic acid (CS2); methyl 3,11-dioxolean-12-ene-28-oleate (DIOXOL); ZCVI4-2; benzyl 3-dehydro-oxy-1,2,5-oxadiazolo[3',4':2,3]oleanolate);The method is selected from the group consisting of a left ventricular assist device (LVAD) or a lung and / or heart transplant. In some embodiments, the patient is treated with one or more agents selected from the group consisting of phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulants, prostacyclin receptor agonists, and endothelin receptor antagonists. In some embodiments, one or more agents are selected from the group consisting of bosentan, sildenafil, beraprost, macitentan, selexipag, epoprostenol, treprostinil, iloprost, ambrisentan, and tadalafil. In some embodiments, the method further comprises the administration of one or more vasodilators. In some embodiments, the method further comprises the administration of one or more agents selected from the group consisting of phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulants, prostacyclin receptor agonists, and endothelin receptor antagonists. In some embodiments, one or more agents are selected from the group consisting of bosentan, sildenafil, beraprost, macitentan, selexipag, epoprostenol, treprostinil, iloprost, ambrisentan, and tadalafil. In some embodiments, the patient was treated with one or more vasodilators before the administration of the polypeptide. In some embodiments, the method further comprises the administration of one or more vasodilators. In some embodiments, one or more vasodilators are selected from the group consisting of prostacyclin, epoprostenol, and sildenafil. In some embodiments, the vasodilator is prostacyclin.

[0019] In some embodiments, the patient receives one or more therapies for PAH. In some embodiments, one or more therapies for PAH include: prostacyclins and their derivatives (e.g., epoprostenol, treprostinil, and iloprost); prostacyclin receptor agonists (e.g., selexipag); endothelin receptor antagonists (e.g., therin, ambrisentan, macitentan, and bosentan); calcium channel blockers (e.g., amlodipine, diltiazem, and nifedipine); anticoagulants (e.g., warfarin); diuretics; oxygen therapy; and atrial septal therapy. Dehistomy; endarterectomy; phosphodiesterase type 5 inhibitors (e.g., sildenafil and tadalafil); soluble guanylate cyclase activators (e.g., synacigat and riociguat); ASK-1 inhibitors (e.g., CIIA; SCH79797; GS-4997; MSC2032964A; 3H-naphtho[1,2,3-de]quinilin-2,7-dione, NQDI-1; 2-thioxothiazolidine, 5-bromo-3-(4-oxo-2-thioxothiazolidine-5-ylidene)-1,3-di Hydro-indole-2-one); NF-κB antagonist (e.g., dh404, CDDO-epoxide; 2,2-difluoropropionamide; C28-imidazole (CDDO-Im); 2-cyano-3,12-dioxolean-1,9-diene-28-euic acid (CDDO); 3-acetyloleanolic acid; 3-trifluoroacetyloleanolic acid; 28-methyl-3-acetyloleanane; 28-methyl-3-trifluoroacetyloleanane; 28-methyloxyoleanolic acid; SZC01 4;SCZ015;SZC017;PEGylated derivatives of oleanolic acid;3-O-(beta-D-glucopyranosyl)oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid;3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester; 3-O-[aL-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid; 3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester; 28-O-β-D-glucopyranosyl-oleanolic acid; 3-O-β- D-glucopyranosyl(1→3)-β-D-glucopyranosidouronic acid (CS1); oleanolic acid 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosidouronic acid (CS2); methyl 3,11-dioxolean-12-ene-28-oleate (DIOXOL); ZCVI4-2; benzyl 3-dehydro-oxy-1,2,5-oxadiazolo[3',4':2,3]oleanolate); selected from a group consisting of a left ventricular assist device (LVAD) or lung and / or heart transplant.

[0020] In certain embodiments, the Disclosure provides a method for treating or preventing cardiopulmonary remodeling associated with pulmonary arterial hypertension in a patient, comprising the step of administering an effective amount of ActRII polypeptide to a patient in need, thereby slowing and / or reversing cardiac remodeling. In some embodiments, the reversal in cardiac remodeling is a sustained reversal. In some embodiments, the cardiopulmonary remodeling is ventricular remodeling. In some embodiments, the ventricular remodeling is left ventricular remodeling. In some embodiments, the ventricular remodeling is right ventricular remodeling. In some embodiments, the cardiopulmonary remodeling is ventricular dilation.

[0021] In certain embodiments, the Disclosure relates to a kit comprising a lyophilized polypeptide and an injection device, wherein the polypeptide begins with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 1, and continues with amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 of SEQ ID NO: 1, The present invention relates to an ActRII polypeptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence ending at any one of 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 99% identical to an amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide consisting of an amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1.In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence corresponding to residues 21-135 of SEQ ID NO: 1. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide is a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Fc domain of the immunoglobulin is the Fc domain of IgG1 immunoglobulin. In some embodiments, the fusion protein further includes a linker domain located between the polypeptide domain and the Fc domain of the immunoglobulin. In some embodiments, the linker domain is selected from the group consisting of TGGG (SEQ ID NO: 20), TGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO: 21). In some embodiments, the linker domain includes TGGG (SEQ ID NO: 20).In some embodiments, the ActRII polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ActRII polypeptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ActRII polypeptide consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the polypeptide is part of a homodimeric protein complex. In some embodiments, the polypeptide is glycosylated. In some embodiments, the polypeptide binds to one or more ligands selected from the group consisting of activin A, activin B, and GDF11. In some embodiments, the polypeptide further binds to one or more ligands selected from the group consisting of BMP10, GDF8, and BMP6. In some embodiments, the polypeptide binds to activin and / or GDF11.

[0022] In some embodiments, the kit comprises one or more vials containing lyophilized polypeptide. In some embodiments, the kit comprises at least two vials containing lyophilized polypeptide. In some embodiments, the two vials may contain the same or different amounts of lyophilized polypeptide. In some embodiments, the vials contain between 25 mg and 60 mg of lyophilized polypeptide. In some embodiments, at least one of the vials contains 60 mg of lyophilized polypeptide. In some embodiments, at least one of the vials contains 45 mg of lyophilized polypeptide. In some embodiments, at least one of the vials contains 30 mg of lyophilized polypeptide. In some embodiments, at least one of the vials contains 25 mg of lyophilized polypeptide. In some embodiments, the first vial contains 45 mg of lyophilized polypeptide and the second vial contains 60 mg of lyophilized polypeptide. In some embodiments, the first vial contains 30 mg of lyophilized polypeptide and the second vial contains 60 mg of lyophilized polypeptide. In some embodiments, the first vial contains 45 mg of lyophilized polypeptide, and the second vial contains 45 mg of lyophilized polypeptide. In some embodiments, the first vial contains 30 mg of lyophilized polypeptide, the second vial contains 45 mg of lyophilized polypeptide, and the third vial contains 60 mg of lyophilized polypeptide. In some embodiments, the first vial contains 25 mg of lyophilized polypeptide, the second vial contains 45 mg of lyophilized polypeptide, and the third vial contains 60 mg of lyophilized polypeptide. In some embodiments, the vials are refrigerated at 2-8°C.

[0023] In some embodiments, the injection device includes a pre-filled syringe. In some embodiments, the injection device includes a pumping device. In some embodiments, the pumping device includes an electromechanical pump assembly. In some embodiments, the pumping device is a wearable pumping device. In some embodiments, the pre-filled syringe includes a reconstitution solution. In some embodiments, the reconstitution solution includes a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier is selected from physiological saline solution, purified water, or sterile water for injection. In some embodiments, the pharmaceutically acceptable excipient is selected from buffers [e.g., citric acid (monohydrate) and / or trisodium citrate (dehydrated)], surfactants (e.g., polysorbate 80), stabilizers (e.g., sucrose), and cryoprotectants (e.g., sucrose). In some embodiments, the injection device includes a vial adapter. In some embodiments, the vial adapter can be attached to a vial. In some embodiments, the vial adapter can be attached to a pre-filled syringe. In some embodiments, pre-filled syringes and vials are attached to both ends of a vial adapter. In some embodiments, the reconstitution solution is transferred from the pre-filled syringe to the vial. In some embodiments, the lyophilized polypeptide is reconstituted into a sterile injection solution. In some embodiments, the lyophilized polypeptide is reconstituted into a sterile injection solution before use. In some embodiments, the sterile injection solution is sterile water for injection. In some embodiments, the sterile injection solution is administered parenterally. In some embodiments, the sterile injection solution is administered via subcutaneous injection. In some embodiments, the sterile injection solution is administered via intradermal injection. In some embodiments, the sterile injection solution is administered via intramuscular injection. In some embodiments, the sterile injection solution is administered via intravenous injection. In some embodiments, the sterile injection solution is self-administered. In some embodiments, an injection device is used to administer the sterile injection solution. In some embodiments, the sterile injection solution contains a therapeutically effective dose. In some embodiments, the therapeutically effective dose contains a dose based on body weight. In some embodiments, the lyophilized polypeptide is administered every three weeks.In some embodiments, the lyophilized polypeptide is administered every four weeks. In some embodiments, the kit is used to treat PAH. In some embodiments, the shelf life of the lyophilized polypeptide is at least 1, 3, 6, 9, or 11 months. In some embodiments, the shelf life of the lyophilized polypeptide is at least 1, 1.5, 2, 2.5, or 3 years. In some embodiments, the lyophilized polypeptide is reconstituted. In some embodiments, the reconstituted polypeptide has a shelf life of at least 2, 3, or 4 hours.

[0024] The patent file contains at least one color drawing / photograph. A copy of the patent containing the color drawing(s) / photograph(s) will be provided by the ministry upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 shows the alignment of the extracellular domains of human ActRIIB (SEQ ID NO: 31) and human ActRIIA (SEQ ID NO: 2), which have residues indicated by boxes that are inferred herein to be in direct contact with the ligand, based on synthetic analysis of multiple ActRIIB and ActRIIA crystal structures.

[0026] [Figure 2] Figure 2 shows the multiple sequence alignment of various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NOs: 6-10 and 36-38).

[0027] [Figure 3]Figure 3 shows the multiple sequence alignment of Fc domains derived from human IgG isotypes using Clustal 2.1. Hinge regions are indicated by dotted underlines. Double underlines indicate positions manipulated in IgG1 Fc (SEQ ID NO: 32) to promote asymmetric chain pairing, as well as examples of corresponding positions for other isotypes IgG2 (SEQ ID NO: 33), IgG3 (SEQ ID NO: 34), and IgG4 (SEQ ID NO: 35).

[0028] [Figure 4] Figures 4A and 4B show the purification of ActRIIA-hFc expressed in CHO cells. The purified protein is visualized as a single, clearly defined peak by a sizing column (Figure 4A), and Coomassi-stained SDS-PAGE (Figure 4B) (left lane: molecular weight standard; right lane: ActRIIA-hFc).

[0029] [Figure 5] Figures 5A and 5B show the binding of ActRIIA-hFc to activin (Figure 5A) and GDF-11 (Figure 5B) as measured by the Biacore® assay.

[0030] [Figure 6] Figure 6 shows the effects of sildenafil and ActRIIA-mFc treatment as mediators on vasomuscularization in a monocothalin rat model of pulmonary arterial hypertension.

[0031] [Figure 7] Figure 7 shows the effects of the vehicle, sildenafil, and ActRIIA-mFc treatment on vasomuscularization in the Sugen hypoxic rat model of pulmonary arterial hypertension.

[0032] [Figure 8A-B]Figures 8A–8D show the changes in pulmonary vascular resistance from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 8A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in pulmonary vascular resistance compared to placebo was -145.8 dyne-seconds / cm5 (95% CI, -241.0 to -50.6) for sotatercept 0.3 mg / kg and -239.5 dyne-seconds / cm5 (95% CI, -329.3 to -149.7) for sotatercept 0.7 mg / kg. Figure 8B shows the change in pulmonary vascular resistance ± SE between baseline and the end of the placebo-controlled treatment period (week 24) in the full analysis set for the sotatercept 0.3 mg / kg and 0.7 mg / kg groups. Figure 8C shows the effect of sotatercept 0.3 mg / kg on the change in pulmonary vascular resistance from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using ANOVA with baseline values ​​as a covariance. Figure 8D shows the effect of sotatercept 0.7 mg / kg on the change in pulmonary vascular resistance from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using ANOVA with baseline values ​​as a covariance. [Figure 8C]Figures 8A–8D show the changes in pulmonary vascular resistance from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 8A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in pulmonary vascular resistance compared to placebo was -145.8 dyne-seconds / cm5 (95% CI, -241.0 to -50.6) for sotatercept 0.3 mg / kg and -239.5 dyne-seconds / cm5 (95% CI, -329.3 to -149.7) for sotatercept 0.7 mg / kg. Figure 8B shows the change in pulmonary vascular resistance ± SE between baseline and the end of the placebo-controlled treatment period (week 24) in the full analysis set for the sotatercept 0.3 mg / kg and 0.7 mg / kg groups. Figure 8C shows the effect of sotatercept 0.3 mg / kg on the change in pulmonary vascular resistance from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using ANOVA with baseline values ​​as a covariance. Figure 8D shows the effect of sotatercept 0.7 mg / kg on the change in pulmonary vascular resistance from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using ANOVA with baseline values ​​as a covariance. [Figure 8D]Figures 8A–8D show the changes in pulmonary vascular resistance from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 8A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in pulmonary vascular resistance compared to placebo was -145.8 dyne-seconds / cm5 (95% CI, -241.0 to -50.6) for sotatercept 0.3 mg / kg and -239.5 dyne-seconds / cm5 (95% CI, -329.3 to -149.7) for sotatercept 0.7 mg / kg. Figure 8B shows the change in pulmonary vascular resistance ± SE between baseline and the end of the placebo-controlled treatment period (week 24) in the full analysis set for the sotatercept 0.3 mg / kg and 0.7 mg / kg groups. Figure 8C shows the effect of sotatercept 0.3 mg / kg on the change in pulmonary vascular resistance from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using ANOVA with baseline values ​​as a covariance. Figure 8D shows the effect of sotatercept 0.7 mg / kg on the change in pulmonary vascular resistance from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using ANOVA with baseline values ​​as a covariance.

[0033] [Figure 9A] Figures 9A and 9B show changes in various parameters from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) treatment in patients with pulmonary arterial hypertension. Patients were treated with placebo, sotatercept 0.3 mg / kg, or sotatercept 0.7 mg / kg. [Figure 9B]Figures 9A and 9B show changes in various parameters from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) treatment in patients with pulmonary arterial hypertension. Patients were treated with placebo, sotatercept 0.3 mg / kg, or sotatercept 0.7 mg / kg.

[0034] [Figure 10A] Figures 10A–10C show the change in 6-minute walk distance from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 10A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in 6-minute walk distance compared to placebo was 29.4 m (95% CI, 3.8 to 55.0) for sotatercept 0.3 mg / kg and 21.4 m (95% CI, -2.8 to 45.7) for sotatercept 0.7 mg / kg. Figure 10B shows the effect of sotatercept 0.3 mg / kg on the change in 6-minute walk distance from baseline to week 24 in a patient subgroup. All data are from the full analysis set and compared to placebo using ANOVA with baseline values ​​as covariances. Figure 10C shows the effect of sotatercept 0.7 mg / kg on the change in 6-minute walk distance from baseline to week 24 in a patient subgroup. All data are from the full analysis set and are compared to placebo using analysis of covariance with baseline values ​​as covariates. [Figure 10B]Figures 10A–10C show the change in 6-minute walk distance from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 10A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in 6-minute walk distance compared to placebo was 29.4 m (95% CI, 3.8 to 55.0) for sotatercept 0.3 mg / kg and 21.4 m (95% CI, -2.8 to 45.7) for sotatercept 0.7 mg / kg. Figure 10B shows the effect of sotatercept 0.3 mg / kg on the change in 6-minute walk distance from baseline to week 24 in a patient subgroup. All data are from the full analysis set and compared to placebo using ANOVA with baseline values ​​as covariances. Figure 10C shows the effect of sotatercept 0.7 mg / kg on the change in 6-minute walk distance from baseline to week 24 in a patient subgroup. All data are from the full analysis set and are compared to placebo using analysis of covariance with baseline values ​​as covariates. [Figure 10C]Figures 10A–10C show the change in 6-minute walk distance from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 10A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in 6-minute walk distance compared to placebo was 29.4 m (95% CI, 3.8 to 55.0) for sotatercept 0.3 mg / kg and 21.4 m (95% CI, -2.8 to 45.7) for sotatercept 0.7 mg / kg. Figure 10B shows the effect of sotatercept 0.3 mg / kg on the change in 6-minute walk distance from baseline to week 24 in a patient subgroup. All data are from the full analysis set and compared to placebo using ANOVA with baseline values ​​as covariances. Figure 10C shows the effect of sotatercept 0.7 mg / kg on the change in 6-minute walk distance from baseline to week 24 in a patient subgroup. All data are from the full analysis set and are compared to placebo using analysis of covariance with baseline values ​​as covariates.

[0035] [Figure 11A]Figures 11A–11C show the change in NT-proBNP from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 11A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in NT-ProBNP compared to placebo was -931.5 pg / mL (95% CI, -1353.24 to -5.0.70) for sotatercept 0.3 mg / kg and -651.0 pg / mL (95% CI, -1043.28 to -258.74) for sotatercept 0.7 mg / kg. Figure 11B shows the effect of sotatercept 0.3 mg / kg on the change in NT-proBNP from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using analysis of covariance with baseline values ​​as covariances. Figure 11C shows the effect of sotatercept 0.7 mg / kg on the change in NT-proBNP from baseline to week 24 in a patient subgroup. All data are derived from the full analysis set and compared to placebo using analysis of covariance with baseline values ​​as covariances. [Figure 11B]Figures 11A–11C show the change in NT-proBNP from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 11A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in NT-ProBNP compared to placebo was -931.5 pg / mL (95% CI, -1353.24 to -5.0.70) for sotatercept 0.3 mg / kg and -651.0 pg / mL (95% CI, -1043.28 to -258.74) for sotatercept 0.7 mg / kg. Figure 11B shows the effect of sotatercept 0.3 mg / kg on the change in NT-proBNP from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using analysis of covariance with baseline values ​​as covariances. Figure 11C shows the effect of sotatercept 0.7 mg / kg on the change in NT-proBNP from baseline to week 24 in a patient subgroup. All data are derived from the full analysis set and compared to placebo using analysis of covariance with baseline values ​​as covariances. [Figure 11C]Figures 11A–11C show the change in NT-proBNP from baseline to week 24 in a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 11A shows the least squares mean ± SE in the full analysis set. The least squares mean difference in NT-ProBNP compared to placebo was -931.5 pg / mL (95% CI, -1353.24 to -5.0.70) for sotatercept 0.3 mg / kg and -651.0 pg / mL (95% CI, -1043.28 to -258.74) for sotatercept 0.7 mg / kg. Figure 11B shows the effect of sotatercept 0.3 mg / kg on the change in NT-proBNP from baseline to week 24 in patient subgroups. All data are derived from the full analysis set and compared to placebo using analysis of covariance with baseline values ​​as covariances. Figure 11C shows the effect of sotatercept 0.7 mg / kg on the change in NT-proBNP from baseline to week 24 in a patient subgroup. All data are derived from the full analysis set and compared to placebo using analysis of covariance with baseline values ​​as covariances.

[0036] [Figure 12]Figure 12 shows the mean changes in echocardiographic parameters from baseline to week 24 during a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) treatment in patients with pulmonary arterial hypertension. Baseline data are mean (SD), and changes are mean LS (SE) derived from an evaluable analysis set. All echocardiographic data were obtained in 2D. TAPSE: tricuspid annular plane systolic excursion; RVFAC: fractional area change. Week 24 includes the end-of-treatment visit if the subject discontinued treatment prior to the week 24 visit. P-values ​​are based on ANCOVA analysis with baseline WHO function class and baseline results as covariates.

[0037] [Figure 13A-C]Figures 13A–13F show that the sotatercept analog RAP-011 (ActRIIA-mFc polypeptide) prevents hypertension (PH) and reduces right ventricular hypertrophy in a mouse model of BMPR2 deficiency. The experimental strategy used to test the prophylactic effect of RAP-011 in mice with Bmpr2 haploinsufficiency is described below. Bmpr2+ / R899X mice were exposed to normobaric hypoxia (FiO2=0.10) and treated twice weekly with either RAP-011 (10 mg / kg, sc) or a medium (PBS) for 5 weeks (Figure 13A). Figure 13B shows right ventricular systolic pressure (RVSP), and Figure 13C shows the Fulton index calculated as the ratio of right ventricular mass (RV) to combined left ventricular and septal mass (LV+S). Data are mean ± SEM (n=7–10 per group). Analysis by one-way ANOVA and Tukey post-hoc test. (Figure 13D shows that amplification of genomic DNA by PCR and direct sequencing confirmed the presence of heterozygous mutations (arrows) in Bmpr2+ / R899X mice (equal peak heights for wild-type and mutant alleles). Figure 13E shows immunoblots of lung homogenates from wild-type and Bmpr2+ / R899X mice analyzed to determine BMPR2 expression. Figure 13F shows quantification of BMPR2 protein expression normalized to GADPH. Data are mean ± SEM (n=5 per group). Analysis by Student's t-test. *P<0.05, ***P<0.001, ****P<0.0001.) [Figure 13D-F]Figures 13A–13F show that the sotatercept analog RAP-011 (ActRIIA-mFc polypeptide) prevents hypertension (PH) and reduces right ventricular hypertrophy in a mouse model of BMPR2 deficiency. The experimental strategy used to test the prophylactic effect of RAP-011 in mice with Bmpr2 haploinsufficiency is described below. Bmpr2+ / R899X mice were exposed to normobaric hypoxia (FiO2=0.10) and treated twice weekly with either RAP-011 (10 mg / kg, sc) or a medium (PBS) for 5 weeks (Figure 13A). Figure 13B shows right ventricular systolic pressure (RVSP), and Figure 13C shows the Fulton index calculated as the ratio of right ventricular mass (RV) to combined left ventricular and septal mass (LV+S). Data are mean ± SEM (n=7–10 per group). Analysis by one-way ANOVA and Tukey post-hoc test. (Figure 13D shows that amplification of genomic DNA by PCR and direct sequencing confirmed the presence of heterozygous mutations (arrows) in Bmpr2+ / R899X mice (equal peak heights for wild-type and mutant alleles). Figure 13E shows immunoblots of lung homogenates from wild-type and Bmpr2+ / R899X mice analyzed to determine BMPR2 expression. Figure 13F shows quantification of BMPR2 protein expression normalized to GADPH. Data are mean ± SEM (n=5 per group). Analysis by Student's t-test. *P<0.05, ***P<0.001, ****P<0.0001.)

[0038] [Figure 14A-C]Figures 14A–14E demonstrate that RAP-011 is effective in combination and monotherapy to reverse pulmonary vascular remodeling in severe experimental PAH. Figure 14A shows the experimental strategy used to test the therapeutic effect of RAP-011 in a Sugen-hypoxic-oxygenous (SuHxNx) rat model of severe PAH. Rats were treated with a single dose of SU5416 (20 mg / kg) on ​​day 0 and exposed to normobaric hypoxia (FiO2=0.10) for 3 weeks, followed by 6 weeks of oxygenous exposure to induce disease progression. The rats were then treated with RAP-011 (2.5 mg / kg, sc, twice weekly), sildenafil (30 mg / kg, po, twice daily), combination therapy with RAP-011 and sildenafil, or a medium (PBS) for 4 weeks, starting 5 weeks after SU5416. Figure 14B shows RVSP, and Figure 14C shows Total Pulmonary Resistance Index (TPRI). Data are mean ± SEM (n=7-14 per group). Figure 14D shows images of representative lung sections stained with hematoxylin and eosin. Scale bar, 200 μm. Figure 14E shows images of lung sections immunostained with antibody against α-smooth muscle actin to illustrate the grade of lung histopathology. Scale bar, 50 μm. Percentage of pulmonary artery vessels classified as Grade 0 (normal, no occlusion), Grade 1 (<50% occlusion), or Grade 2 (>50% occlusion), grouped according to vascular outer diameter. Data are mean ± SEM (n=4 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test; for simplification, only significance for the percentage of Grade 0 vessels is indicated (*P<0.05). [Figure 14D]Figures 14A–14E demonstrate that RAP-011 is effective in combination and monotherapy to reverse pulmonary vascular remodeling in severe experimental PAH. Figure 14A shows the experimental strategy used to test the therapeutic effect of RAP-011 in a Sugen-hypoxic-oxygenous (SuHxNx) rat model of severe PAH. Rats were treated with a single dose of SU5416 (20 mg / kg) on ​​day 0 and exposed to normobaric hypoxia (FiO2=0.10) for 3 weeks, followed by 6 weeks of oxygenous exposure to induce disease progression. The rats were then treated with RAP-011 (2.5 mg / kg, sc, twice weekly), sildenafil (30 mg / kg, po, twice daily), combination therapy with RAP-011 and sildenafil, or a medium (PBS) for 4 weeks, starting 5 weeks after SU5416. Figure 14B shows RVSP, and Figure 14C shows Total Pulmonary Resistance Index (TPRI). Data are mean ± SEM (n=7-14 per group). Figure 14D shows images of representative lung sections stained with hematoxylin and eosin. Scale bar, 200 μm. Figure 14E shows images of lung sections immunostained with antibody against α-smooth muscle actin to illustrate the grade of lung histopathology. Scale bar, 50 μm. Percentage of pulmonary artery vessels classified as Grade 0 (normal, no occlusion), Grade 1 (<50% occlusion), or Grade 2 (>50% occlusion), grouped according to vascular outer diameter. Data are mean ± SEM (n=4 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test; for simplification, only significance for the percentage of Grade 0 vessels is indicated (*P<0.05). [Figure 14E]Figures 14A–14E demonstrate that RAP-011 is effective in combination and monotherapy to reverse pulmonary vascular remodeling in severe experimental PAH. Figure 14A shows the experimental strategy used to test the therapeutic effect of RAP-011 in a Sugen-hypoxic-oxygenous (SuHxNx) rat model of severe PAH. Rats were treated with a single dose of SU5416 (20 mg / kg) on ​​day 0 and exposed to normobaric hypoxia (FiO2=0.10) for 3 weeks, followed by 6 weeks of oxygenous exposure to induce disease progression. The rats were then treated with RAP-011 (2.5 mg / kg, sc, twice weekly), sildenafil (30 mg / kg, po, twice daily), combination therapy with RAP-011 and sildenafil, or a medium (PBS) for 4 weeks, starting 5 weeks after SU5416. Figure 14B shows RVSP, and Figure 14C shows Total Pulmonary Resistance Index (TPRI). Data are mean ± SEM (n=7-14 per group). Figure 14D shows images of representative lung sections stained with hematoxylin and eosin. Scale bar, 200 μm. Figure 14E shows images of lung sections immunostained with antibody against α-smooth muscle actin to illustrate the grade of lung histopathology. Scale bar, 50 μm. Percentage of pulmonary artery vessels classified as Grade 0 (normal, no occlusion), Grade 1 (<50% occlusion), or Grade 2 (>50% occlusion), grouped according to vascular outer diameter. Data are mean ± SEM (n=4 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test; for simplification, only significance for the percentage of Grade 0 vessels is indicated (*P<0.05).

[0039] [Figure 15A-D]Figures 15A–15G show that the simultaneous inhibition of activin, GDF8, and GDF11 contributes to the effect of RAP-011 in vitro and in vivo in the PH model. Figure 15A shows the effect of therapeutic treatment with RAP-011 on lung cell proliferation in the SuHxNx model of severe PAH, measured by the percentage of Ki67-positive cells. Data are mean ± SEM (n=4–5 rats per group). Figure 15B describes the cell culture system used to investigate the antiproliferative effect of sotatercept. Human pulmonary artery smooth muscle cells (PASMCs) were treated in conditioned medium collected from human pulmonary artery endothelial cells (PAECs) in the absence or presence of separate antibodies against activin A and activin B (anti-Act), dual antibodies against GDF8 and GDF11 (anti-GDF), combined anti-Act and anti-GDF, or sotatercept (ACE-011). As shown in Figure 15C, PASMC proliferation was quantified in the bromodeoxyuridine (BrdU) assay. Data are mean ± SEM (n=8 per group). Figure 15D illustrates the experimental strategy used to test the prophylactic effect of multiligand inhibition in a SuHx rat model of PH. Rats were treated with a single dose of SU5416 (20 mg / kg, sc), exposed to normobaric hypoxia (FiO2=0.13), and treated with anti-Act (10 mg / kg + 10 mg / kg), anti-GDF (10 mg / kg), a combination of anti-Act and anti-GDF, or sc twice a week for 4 weeks, starting 1 day after SU5416. Figure 15E shows systolic pulmonary artery pressure (sPAP) in rats treated according to Figure 15D. Figure 15F shows mPAP in rats treated according to Figure 15D. Figure 15G shows the Fulton index in rats treated according to Figure 15D. Data are mean ± SEM (n=5-9 rats per group). Analysis was performed using one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ****P<0.0001). [Figure 15E-G]Figures 15A–15G show that the simultaneous inhibition of activin, GDF8, and GDF11 contributes to the effect of RAP-011 in vitro and in vivo in the PH model. Figure 15A shows the effect of therapeutic treatment with RAP-011 on lung cell proliferation in the SuHxNx model of severe PAH, measured by the percentage of Ki67-positive cells. Data are mean ± SEM (n=4–5 rats per group). Figure 15B describes the cell culture system used to investigate the antiproliferative effect of sotatercept. Human pulmonary artery smooth muscle cells (PASMCs) were treated in conditioned medium collected from human pulmonary artery endothelial cells (PAECs) in the absence or presence of separate antibodies against activin A and activin B (anti-Act), dual antibodies against GDF8 and GDF11 (anti-GDF), combined anti-Act and anti-GDF, or sotatercept (ACE-011). As shown in Figure 15C, PASMC proliferation was quantified in the bromodeoxyuridine (BrdU) assay. Data are mean ± SEM (n=8 per group). Figure 15D illustrates the experimental strategy used to test the prophylactic effect of multiligand inhibition in a SuHx rat model of PH. Rats were treated with a single dose of SU5416 (20 mg / kg, sc), exposed to normobaric hypoxia (FiO2=0.13), and treated with anti-Act (10 mg / kg + 10 mg / kg), anti-GDF (10 mg / kg), a combination of anti-Act and anti-GDF, or sc twice a week for 4 weeks, starting 1 day after SU5416. Figure 15E shows systolic pulmonary artery pressure (sPAP) in rats treated according to Figure 15D. Figure 15F shows mPAP in rats treated according to Figure 15D. Figure 15G shows the Fulton index in rats treated according to Figure 15D. Data are mean ± SEM (n=5-9 rats per group). Analysis was performed using one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ****P<0.0001).

[0040] [Figure 16A-B]Figures 16A–16K show that therapeutic treatment with RAP-011 instead of sildenafil reduces cardiac hypertrophy, restores septal geometry, and improves right ventricular function in severe experimental PAH. Figure 16A shows the Fulton index in normal or SuHxNx rats as a function of treatment, and Figure 16B shows the cardiac index (CI). Data are mean ± SEM (n=7–13 rats per group). Figure 16C shows representative echocardiographic images obtained in a replicated manner from individual SuHxNx rats before and after therapy. Figure 16D shows pulmonary artery acceleration time (PAAT) in animals treated according to Example 13. Figure 16E shows tricuspid annular systolic displacement (TAPSE) in animals treated according to Example 13. Figure 16F shows right ventricular wall thickness (RVWT) measured in diastole in animals treated according to Example 13. Figure 16G shows the percentage change in right ventricular area (RVFAC) in animals treated according to Example 13. Data are mean ± SEM (n=7–11 rats per group). Figures 16H–16K show the ratio of myosin heavy chain isoform expression (Myh7:Myh6) (Figure 16H) as a function of treatment in the right ventricle of normal or SuHxNx rats, as well as the levels of Nppb (Figure 16I), Inhba (Figure 16J), and Inhbb (Figure 16K) mRNA. Data are mean ± SEM (n=6–11 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 16C]Figures 16A–16K show that therapeutic treatment with RAP-011 instead of sildenafil reduces cardiac hypertrophy, restores septal geometry, and improves right ventricular function in severe experimental PAH. Figure 16A shows the Fulton index in normal or SuHxNx rats as a function of treatment, and Figure 16B shows the cardiac index (CI). Data are mean ± SEM (n=7–13 rats per group). Figure 16C shows representative echocardiographic images obtained in a replicated manner from individual SuHxNx rats before and after therapy. Figure 16D shows pulmonary artery acceleration time (PAAT) in animals treated according to Example 13. Figure 16E shows tricuspid annular systolic displacement (TAPSE) in animals treated according to Example 13. Figure 16F shows right ventricular wall thickness (RVWT) measured in diastole in animals treated according to Example 13. Figure 16G shows the percentage change in right ventricular area (RVFAC) in animals treated according to Example 13. Data are mean ± SEM (n=7–11 rats per group). Figures 16H–16K show the ratio of myosin heavy chain isoform expression (Myh7:Myh6) (Figure 16H) as a function of treatment in the right ventricle of normal or SuHxNx rats, as well as the levels of Nppb (Figure 16I), Inhba (Figure 16J), and Inhbb (Figure 16K) mRNA. Data are mean ± SEM (n=6–11 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 16D-G]Figures 16A–16K show that therapeutic treatment with RAP-011 instead of sildenafil reduces cardiac hypertrophy, restores septal geometry, and improves right ventricular function in severe experimental PAH. Figure 16A shows the Fulton index in normal or SuHxNx rats as a function of treatment, and Figure 16B shows the cardiac index (CI). Data are mean ± SEM (n=7–13 rats per group). Figure 16C shows representative echocardiographic images obtained in a replicated manner from individual SuHxNx rats before and after therapy. Figure 16D shows pulmonary artery acceleration time (PAAT) in animals treated according to Example 13. Figure 16E shows tricuspid annular systolic displacement (TAPSE) in animals treated according to Example 13. Figure 16F shows right ventricular wall thickness (RVWT) measured in diastole in animals treated according to Example 13. Figure 16G shows the percentage change in right ventricular area (RVFAC) in animals treated according to Example 13. Data are mean ± SEM (n=7–11 rats per group). Figures 16H–16K show the ratio of myosin heavy chain isoform expression (Myh7:Myh6) (Figure 16H) as a function of treatment in the right ventricle of normal or SuHxNx rats, as well as the levels of Nppb (Figure 16I), Inhba (Figure 16J), and Inhbb (Figure 16K) mRNA. Data are mean ± SEM (n=6–11 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 16H-K]Figures 16A–16K show that therapeutic treatment with RAP-011 instead of sildenafil reduces cardiac hypertrophy, restores septal geometry, and improves right ventricular function in severe experimental PAH. Figure 16A shows the Fulton index in normal or SuHxNx rats as a function of treatment, and Figure 16B shows the cardiac index (CI). Data are mean ± SEM (n=7–13 rats per group). Figure 16C shows representative echocardiographic images obtained in a replicated manner from individual SuHxNx rats before and after therapy. Figure 16D shows pulmonary artery acceleration time (PAAT) in animals treated according to Example 13. Figure 16E shows tricuspid annular systolic displacement (TAPSE) in animals treated according to Example 13. Figure 16F shows right ventricular wall thickness (RVWT) measured in diastole in animals treated according to Example 13. Figure 16G shows the percentage change in right ventricular area (RVFAC) in animals treated according to Example 13. Data are mean ± SEM (n=7–11 rats per group). Figures 16H–16K show the ratio of myosin heavy chain isoform expression (Myh7:Myh6) (Figure 16H) as a function of treatment in the right ventricle of normal or SuHxNx rats, as well as the levels of Nppb (Figure 16I), Inhba (Figure 16J), and Inhbb (Figure 16K) mRNA. Data are mean ± SEM (n=6–11 rats per group). Analysis by one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0041] [Figure 17A-D]Figures 17A–17I demonstrate that RAP-011 exerts structural and functional cardioprotective effects in a model of right heart failure induced by pressure overload. Figure 17A shows the experimental strategy used to evaluate the potential cardioprotective effects of RAP-011 in a mouse model of sustained pressure overload. Wild-type mice were subjected to pulmonary artery ligation and treated twice a week for three weeks, starting one day after surgery, with either RAP-011 (10 mg / kg, sc) or the medium (PBS). The various parameters measured using the experimental strategy described in Figure 17A are as follows: Fulton index (Figure 17B), right ventricular free wall thickness (RVFWT) (Figure 17C), TAPSE (Figure 17D), myocardial performance index (MPI) (Figure 17E), right ventricular maximum pressure (RVDP) (Figure 17F), and peak percentages of right ventricular pressure elevation (dP / dtmax) and reduction (-dP / dtmin) (Figure 17G). Data are mean ± SEM (n=10-15 mice per group for day 21). Figure 17H ​​shows a representative image of a right ventricular section stained with Masson's trichrome blue to detect fibrosis (scale bar, 20 μm), and Figure 17I shows the quantification of the percentage area occupied by fibrous tissue. Data are mean ± SEM (n=10-15 mice per group). Analysis was performed using one-way ANOVA and Tukey post-hoc tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 17E-G]Figures 17A–17I demonstrate that RAP-011 exerts structural and functional cardioprotective effects in a model of right heart failure induced by pressure overload. Figure 17A shows the experimental strategy used to evaluate the potential cardioprotective effects of RAP-011 in a mouse model of sustained pressure overload. Wild-type mice were subjected to pulmonary artery ligation and treated twice a week for three weeks, starting one day after surgery, with either RAP-011 (10 mg / kg, sc) or the medium (PBS). The various parameters measured using the experimental strategy described in Figure 17A are as follows: Fulton index (Figure 17B), right ventricular free wall thickness (RVFWT) (Figure 17C), TAPSE (Figure 17D), myocardial performance index (MPI) (Figure 17E), right ventricular maximum pressure (RVDP) (Figure 17F), and peak percentages of right ventricular pressure elevation (dP / dtmax) and reduction (-dP / dtmin) (Figure 17G). Data are mean ± SEM (n=10-15 mice per group for day 21). Figure 17H ​​shows a representative image of a right ventricular section stained with Masson's trichrome blue to detect fibrosis (scale bar, 20 μm), and Figure 17I shows the quantification of the percentage area occupied by fibrous tissue. Data are mean ± SEM (n=10-15 mice per group). Analysis was performed using one-way ANOVA and Tukey post-hoc tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 17H-I]Figures 17A–17I demonstrate that RAP-011 exerts structural and functional cardioprotective effects in a model of right heart failure induced by pressure overload. Figure 17A shows the experimental strategy used to evaluate the potential cardioprotective effects of RAP-011 in a mouse model of sustained pressure overload. Wild-type mice were subjected to pulmonary artery ligation and treated twice a week for three weeks, starting one day after surgery, with either RAP-011 (10 mg / kg, sc) or the medium (PBS). The various parameters measured using the experimental strategy described in Figure 17A are as follows: Fulton index (Figure 17B), right ventricular free wall thickness (RVFWT) (Figure 17C), TAPSE (Figure 17D), myocardial performance index (MPI) (Figure 17E), right ventricular maximum pressure (RVDP) (Figure 17F), and peak percentages of right ventricular pressure elevation (dP / dtmax) and reduction (-dP / dtmin) (Figure 17G). Data are mean ± SEM (n=10-15 mice per group for day 21). Figure 17H ​​shows a representative image of a right ventricular section stained with Masson's trichrome blue to detect fibrosis (scale bar, 20 μm), and Figure 17I shows the quantification of the percentage area occupied by fibrous tissue. Data are mean ± SEM (n=10-15 mice per group). Analysis was performed using one-way ANOVA and Tukey post-hoc tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0042] [Figure 18A-D]Figures 18A–18G demonstrate that the disease reversal effect of RAP-011 persists after discontinuation of treatment in severe experimental PAH. Figure 18A shows the experimental strategy used to test the persistence of the therapeutic effect of RAP-011 in a SuHxNx rat model of severe PAH. Rats were treated with a single dose of SU5416 (20 mg / kg, sc) on day 0, followed by 3 weeks of normobaric hypoxia (FiO2=0.10), and then 10 weeks of normal oxygenation to induce disease progression. The rats were then treated twice a week with RAP-011 (2.5 mg / kg, sc) or a medium (PBS) from week 5 to week 9 after SU5416, with treatment discontinued at week 9 for the remaining 4 weeks. The following parameters were determined as functions of treatment: RVSP (Figure 18B), TPRI (Figure 18C), Fulton index (Figure 18D), CI (Figure 18E), PAAT (Figure 18F), and TAPSE (Figure 18G). Data are mean ± SEM (n=7–13 rats per group). Analysis was performed using one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ****P<0.0001). [Figure 18E-G] Figures 18A–18G demonstrate that the disease reversal effect of RAP-011 persists after discontinuation of treatment in severe experimental PAH. Figure 18A shows the experimental strategy used to test the persistence of the therapeutic effect of RAP-011 in a SuHxNx rat model of severe PAH. Rats were treated with a single dose of SU5416 (20 mg / kg, sc) on day 0, followed by 3 weeks of normobaric hypoxia (FiO2=0.10), and then 10 weeks of normal oxygenation to induce disease progression. The rats were then treated twice a week with RAP-011 (2.5 mg / kg, sc) or a medium (PBS) from week 5 to week 9 after SU5416, with treatment discontinued at week 9 for the remaining 4 weeks. The following parameters were determined as functions of treatment: RVSP (Figure 18B), TPRI (Figure 18C), Fulton index (Figure 18D), CI (Figure 18E), PAAT (Figure 18F), and TAPSE (Figure 18G). Data are mean ± SEM (n=7–13 rats per group). Analysis was performed using one-way ANOVA and Tukey post-hoc test (*P<0.05, **P<0.01, ****P<0.0001).

[0043] [Figure 19] Figure 19 shows the components of the kit, including lyophilized polypeptide and an injection device. A vial (1) holds lyophilized polypeptide, reconstituted sterile injection solution, or sterile injection solution. From (1) is a pre-filled syringe (2) containing a reconstitution solution used to reconstitute the lyophilized polypeptide into a sterile injection solution. A vial adapter (3) connects vial (1) to pre-filled syringe (2) by attaching to the vial at one end and to the pre-filled syringe at both ends. A syringe (4) and needle (5) are provided for administering the sterile injection solution. A swab material (6) is provided for sterilizing the individual kit components.

[0044] [Figure 20A-C] Figures 20A–20C show the mean changes in echocardiographic parameters from baseline to week 24 during a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 20A shows the improvement in mean LS (SE) change of right ventricular end-diastolic area (RVEDA) from baseline to week 24. Figure 20B shows the improvement in mean LS (SE) change of right ventricular end-systolic area (RVESA) from baseline to week 24. Figure 20C presents the mean LS and p-value data derived from Figures 20A and 20B in tabular format. Baseline data are mean (SD), and changes are mean LS (SE) derived from the evaluable analysis set. All echocardiographic data were obtained in 2D. Bar graphs represent mean ± standard deviation. †EOP represents data obtained at the end of the placebo-controlled treatment period (week 24). ‡Standard errors are shown in parentheses relative to the overall LS mean. CI: Confidence Interval; EOP: End of Placebo-Controlled Treatment Period; LS: Least Squares; SOC: Standard of Care.

[0045] [Figure 21A-B]Figures 21A and 21B show the mean change in pulmonary artery systolic pressure (PASP) from baseline to week 24, as measured during a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) in patients with pulmonary arterial hypertension. Figure 21A shows the improvement in pulmonary artery systolic pressure (PASP) in patients treated with 0.3 mg / kg sotatercept and standard care (SOC) or 0.7 mg / kg sotatercept and SOC. Figure 21B presents the mean LS and p-value data derived from Figure 21A in tabular format. Baseline data are mean (SD), and change is mean LS (SE) derived from the evaluable analysis set. All echocardiographic data were obtained in 2D. Bar graphs represent mean ± standard deviation. †EOP represents data obtained at the end of the placebo-controlled treatment period (week 24). ‡Standard error is shown in parentheses relative to the overall mean LS. CI: Confidence interval; LS: Least squares.

[0046] [Figure 22A-B] Figures 22A and 22B show the mean change in right ventricular-pulmonary (RV-PA) coupling from baseline to week 24 as measured during a placebo-controlled clinical trial using sotatercept (ActRIIA-hFc polypeptide) treatment in patients with pulmonary arterial hypertension. Figure 22A shows improvement in RV-PA coupling in patients treated with 0.3 mg / kg sotatercept and standard care (SOC) or 0.7 mg / kg sotatercept and SOC. Figure 22B presents the mean LS and p-value data derived from Figure 22A in tabular format. Baseline data are mean (SD), and change is mean LS (SE) derived from the evaluable analysis set. All echocardiographic data were obtained in 2D. Bar graphs represent mean ± standard deviation. †EOP represents data obtained at the end of the placebo-controlled treatment period (week 24). ‡Standard error is shown in parentheses relative to the overall mean LS. §The cutoff value for RV-PA coupling has not been validated in large cohorts. CI: Confidence interval; LS: Least squares; Values ​​in parentheses are relative to the overall LS mean; RV-PA: Right ventricle-pulmonary artery.

[0047] [Figure 23A-C] Figures 23A to 23F show that treatment with ActRIIA-mFc polypeptide prevents PH and reduces right ventricular hypertrophy in a mouse model of BMPR2 haploinsufficiency. The experimental strategy used to test the prophylactic effect of ActRIIA-mFc in a mouse model of Bmpr2 haploinsufficiency is shown in Figure 23A. Twenty-nine Bmpr2+ / R899X mice were randomized into three groups: (i) seven mice were housed under normal oxygen pressure conditions for five weeks, "Nx"; (ii) eleven mice were housed under hypoxic conditions and subcutaneously injected with a medium control (phosphate-buffered saline (PBS)) twice a week for five weeks, "Hx Veh"; and (iii) eleven mice were housed under hypoxic conditions and subcutaneously injected with ActRIIA-mFc at a dose of 10 mg / kg twice a week for five weeks, "Hx ActRIIA-mFc". Figure 23B shows pulmonary artery acceleration time (PAAT), and Figure 23C shows right ventricular systolic pressure (RVSP). Figure 23D shows right ventricular free wall thickness (RVWT), and Figure 23E shows the Fulton index calculated as the ratio of right ventricular weight (RV) to the combined weight of the left ventricle and septum (LV+S) (RV / (LV+S)). Figure 23F shows tricuspid annular systolic displacement (TAPSE). Data are mean ± SEM (n=7-11 per group). Analysis was performed using one-way ANOVA and Tukey post-hoc test. *P<0.05, ***P<0.001, ****P<0.0001. [Figure 23D-F]Figures 23A to 23F show that treatment with ActRIIA-mFc polypeptide prevents PH and reduces right ventricular hypertrophy in a mouse model of BMPR2 haploinsufficiency. The experimental strategy used to test the prophylactic effect of ActRIIA-mFc in a mouse model of Bmpr2 haploinsufficiency is shown in Figure 23A. Twenty-nine Bmpr2+ / R899X mice were randomized into three groups: (i) seven mice were housed under normal oxygen pressure conditions for five weeks, "Nx"; (ii) eleven mice were housed under hypoxic conditions and subcutaneously injected with a medium control (phosphate-buffered saline (PBS)) twice a week for five weeks, "Hx Veh"; and (iii) eleven mice were housed under hypoxic conditions and subcutaneously injected with ActRIIA-mFc at a dose of 10 mg / kg twice a week for five weeks, "Hx ActRIIA-mFc". Figure 23B shows pulmonary artery acceleration time (PAAT), and Figure 23C shows right ventricular systolic pressure (RVSP). Figure 23D shows right ventricular free wall thickness (RVWT), and Figure 23E shows the Fulton index calculated as the ratio of right ventricular weight (RV) to the combined weight of the left ventricle and septum (LV+S) (RV / (LV+S)). Figure 23F shows tricuspid annular systolic displacement (TAPSE). Data are mean ± SEM (n=7-11 per group). Analysis was performed using one-way ANOVA and Tukey post-hoc test. *P<0.05, ***P<0.001, ****P<0.0001.

[0048] [Figure 24A-B]Figures 24A and 24B show that treatment with ActRIIA-mFc polypeptide prevents perivascular inflammation by preventing macrophage infiltration in the lungs. Twenty-nine Bmpr2+ / R899X mice were randomized into three groups: (i) seven mice were housed under normal oxygen pressure conditions for five weeks, designated "Nx"; (ii) eleven mice were housed under hypoxic conditions and subcutaneously injected with a medium control (phosphate-buffered saline (PBS)) twice a week for five weeks, designated "Hx Veh"; and (iii) eleven mice were housed under hypoxic conditions and subcutaneously injected with ActRIIA-mFc at a dose of 10 mg / kg twice a week for five weeks, designated "Hx ActRIIA-mFc". Figure 24A shows postmortem analysis of macrophage infiltration in the lungs by immunohistochemical staining for the macrophage marker F4 / 80. Figure 24B shows the quantification of the percentage of F4 / 80 positive cells in the lungs based on the evaluation of 40 high-magnification fields per animal. Data are mean ± SEM (n=7-11 per group). Analysis by one-way ANOVA and Tukey post-hoc test. *P<0.05, ***P<0.001, ****P<0.0001. [Modes for carrying out the invention]

[0049] Detailed explanation 1. Overview This disclosure relates to compositions for treating pulmonary arterial hypertension (e.g., class II or class III of function) and methods comprising the step of administering an effective amount of the ActRII polypeptide described herein to a patient in need. In certain embodiments, this disclosure provides a method for treating or preventing pulmonary arterial hypertension in an individual in need by administering to the individual a therapeutically effective amount of the ActRII polypeptide described herein.

[0050] Pulmonary arterial hypertension (PH) [WHO Group 1 pulmonary hypertension] is a severe, progressive, and life-threatening disease of the pulmonary vascular structure characterized by severe narrowing of blood vessels and abnormal proliferation of smooth muscle cells in the pulmonary artery walls. Severe narrowing of blood vessels in the lungs results in extremely high pulmonary artery pressure. Such high pressure makes it difficult for the heart to pump blood through the lungs for oxygenation. Because the heart struggles to pump against such high pressure, patients with PAH suffer from extreme shortness of breath. Patients with PAH typically develop a significant increase in PVR and a persistent increase in mPAP, which ultimately leads to right ventricular failure and death. Patients diagnosed with PAH have a poor prognosis and a similarly impaired quality of life, with a life expectancy of 2 to 5 years from diagnosis if left untreated.

[0051] PAH can be diagnosed based on mean pulmonary artery pressure (PHA) above 25 mmHg at rest (or above 20 mmHg according to the latest updated guidelines) and normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, syncope, and other symptoms, all of which are aggravated by exertion. PAH can be a severe disease with significantly reduced exercise tolerance and heart failure. There are two main types of PAH: idiopathic PAH (e.g., PAH with no identified etiology) and hereditary PAH (e.g., PAH associated with mutations in BMPR2, ALK1, ENG, SMAD9, CAV1, KCNK3, or EIF2AK4). In 70% of familial PAH cases, the mutation is located in the BMPR2 gene. Risk factors for developing PAH include a family history of PAH, drug and toxin use (e.g., methamphetamine or cocaine use), infections (e.g., HIV infection or schistosomiasis), cirrhosis of the liver, congenital cardiac anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus). PAH may be associated with long-term responses to calcium channel blockers, a clear feature of venous / capillary (PVOD / PCH) involvement, and persistent PH in neonatal syndromes.

[0052] The terms used herein generally have their common meanings in the art within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners when describing the compositions and methods of this disclosure and how they are prepared and used. The scope or meaning of any use of a term will become clear from the specific context in which it is used.

[0053] The term "sequence similarity" refers to the degree of identity or agreement between nucleic acid sequences or amino acid sequences that may or may not share a common evolutionary origin in all their grammatical forms.

[0054] "Sequence identity percentage (%)" is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to amino acid residues (or nucleic acids) in a reference polypeptide (or nucleotide) sequence, after aligning the sequence with respect to a reference polypeptide (or nucleotide) sequence and, if necessary, introducing gaps to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways within the scope of the skill of the art, for example, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, ALIGN-2, Clustal Omega, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. In some embodiments, the value of amino acid (nucleic acid) sequence identity % is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX® operating systems, including Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.Other algorithms for determining sequence identity or homology include: Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ), LALIGN (http: / / www.ebi.ac.uk / Tools / psa / lalign / and http: / / www.ebi.ac.uk / Tools / psa / lalign / nucleotide.html), FASTA (http: / / www.ebi.ac.uk / Tools / sss / fasta / ), SIM (http: / / web.expasy.org / sim / ), and EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). In preferred embodiments, the algorithm used to determine sequence identity is Clustal Omega.

[0055] In all its grammatical forms, "agonize" refers to the process of activating a protein and / or gene (for example, by activating or amplifying the gene expression of that protein, or by inducing an inactive protein to become active), or by increasing the activity of a protein and / or gene.

[0056] "Antagonizing," in all its grammatical forms, refers to the process of inhibiting a protein and / or gene (for example, by inhibiting or reducing the gene expression of that protein, or by inducing an active protein to become inactive), or by reducing the activity of a protein and / or gene.

[0057] When the terms “about” and “approximately” are used in relation to numbers throughout this specification and the claims, they represent an interval of precision that is familiar to and acceptable to those skilled in the art. Generally, such an interval of precision is ±10%. Alternatively, and especially in biological systems, the terms “about” and “approximately” may mean a value that is within one decimal place of a given value, preferably ≤5 times, and more preferably ≤2 times.

[0058] Numerical ranges disclosed herein include numbers that define the range.

[0059] The terms “one (a)” and “one (an)” include multiple referents unless the context in which the terms are used explicitly indicates otherwise. The terms “one (a)” (or “one (an)”), as well as “one or more” and “at least one” can be used interchangeably herein. Furthermore, “and / or” should be considered, when used herein, as a specific disclosure of each of two or more designated characteristics or components, with or without the other. Thus, when the term “and / or” is used herein in phrases such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0060] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to mean the inclusion of the integer or group of integers mentioned, but not the exclusion of any other integer or group of integers.

[0061] 2. ActRII polypeptide In certain aspects, this disclosure relates to ActRII polypeptides and their use (e.g., to treat, prevent, or reduce the rate of progression and / or severity of pulmonary arterial hypertension or one or more complications thereof). As used herein, the term “ActRII” refers to the family of type II activin receptors, which include activin receptor type IIA (ActRIIA) and activin receptor type IIB (ActRIIB).

[0062] In certain embodiments, the disclosure relates to an ActRII polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 23, 27, 30, and 41. In other embodiments, the disclosure relates to an ActRII polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 31. As used herein, the term “ActRII” refers to the family of activin receptor type IIA (ActRIIA) proteins, the family of activin receptor type IIB (ActRIIB) proteins, or combinations thereof and / or variants. ActRII polypeptides may originate from any species and may include variants derived from such ActRII proteins by mutagenesis or other modifications. References to ActRII herein are understood to refer to any one of the currently identified forms. Members of the ActRII family are generally transmembrane proteins comprising a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain having putative serine / threonine kinase activity.

[0063] The term ActRII polypeptide includes any naturally occurring polypeptide of an ActRII family member, and any variant thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such variant ActRII polypeptides are provided throughout this disclosure and in International Patent Application Publication Nos. WO2006 / 012627, WO2007 / 062188, WO2008 / 097541, WO2010 / 151426, and WO2011 / 020045, which are incorporated herein by reference in their entirety. The amino acid numbering for all ActRII-related polypeptides described herein is based on the numbering of the human ActRII precursor protein sequence (SEQ ID NO: 1) provided below, unless otherwise specifically specified.

[0064] The standard human ActRII precursor protein sequence is as follows: [ka]

[0065] Signal peptides are indicated by a single underline; extracellular domains are indicated in bold font; and possible endogenous N-linked glycosylation sites are indicated by a double underline.

[0066] The processed (mature) extracellular human ActRII polypeptide sequence is as follows: [ka]

[0067] The C-terminal "tail" of the extracellular domain is indicated by a single underline. Sequences lacking the "tail" (Δ15 sequences) are as follows: [ka]

[0068] The nucleic acid sequence encoding the human ActRII precursor protein is shown below (SEQ ID NO: 4), and corresponds to nucleotides 159-1700 of the Genbank reference sequence NM_001616.4. The signal sequence is underlined. [ka] [ka]

[0069] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRII polypeptide is as follows: [ka]

[0070] ActRII is well conserved among vertebrates, and the large elongation of the extracellular domain is completely conserved. For example, Figure 2 shows the multiple sequence alignment of the human ActRII extracellular domain compared to various ActRII orthologues. Many of the ligands that bind to ActRII are also highly conserved. Therefore, from these alignments, it is possible to predict not only the critical amino acid positions within the ligand-binding domain that are important for normal ActRII-ligand-binding activity, but also amino acid positions that may tolerate substitution without significantly altering normal ActRII-ligand-binding activity. Thus, an active human ActRII variant polypeptide useful according to the methods disclosed herein may contain one or more amino acids at corresponding positions from the ActRII sequence of another vertebrate, or it may contain residues similar to those in the human or other vertebrate sequence.

[0071] Figure 1 illustrates the amino acid sequence alignment of the human ActRIIA extracellular domain and the human ActRIIB extracellular domain. This alignment shows the amino acid residues in both receptors that are thought to be in direct contact with the ActRII ligand. For example, the composite ActRII structure shows that the ActRIIA-ligand binding pocket is partially defined by residues F31, N33, N35, K38 to T41, E47, Y50, K53 to K55, R57, H58, F60, T62, K74, W78 to N83, Y85, R87, E92, and K94 to F101. Conservative mutations are expected to be tolerated at these positions.

[0072] While not limiting, the following examples illustrate this approach to defining active ActRII variants. As illustrated in Figure 2, F13 in the human extracellular domain is Y in the ActRIIA of Ovis aries (SEQ ID NO: 7), Gallus gallus (SEQ ID NO: 10), Bos Taurus (SEQ ID NO: 36), Tyto alba (SEQ ID NO: 37), and Myotis davidii (SEQ ID NO: 38), indicating that aromatic residues containing F, W, and Y are acceptable at this position. Q24 in the human extracellular domain is R in the ActRIIA of Bos Taurus, indicating that charged residues containing D, R, K, H, and E are acceptable at this position. S95 in the human extracellular domain is F in the ActRIIA of Gallus gallus and Tyto alba, indicating that this site can tolerate a wide variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, and Y, and possibly hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in the ActRIIA of Ovis aries, indicating that acidic residues, including D and E, are tolerated at this position. P29 in the human extracellular domain is relatively unconserved, appearing as S in the ActRIIA of Ovis aries and as L in the ActRIIA of Myotis davidii, thus essentially any amino acid should be tolerated at this position.

[0073] Furthermore, as discussed above, ActRII proteins are characterized in the art in terms of their structural / functional features, particularly with respect to ligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): [1555-1566; and U.S. Patents 7,709,605, 7,612,041, and 7,842,663]. For example, defining a known structural motif as the three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located in variable positions within the extracellular domains of the respective monomeric receptors [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. In addition to the teachings herein, these references provide ample guidance on methods for constructing ActRII variants that retain one or more desired activities (e.g., ligand-binding activity).

[0074] For example, defining a known structural motif as the three-finger toxin fold is crucial for ligand binding by type I and type II receptors, and is formed by conserved cysteine ​​residues located in variable positions within the extracellular domains of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Therefore, the core ligand-binding domain of human ActRII, bounded by the outermost cysteine ​​of these conserved residues, corresponds to positions 30-110 of SEQ ID NO: 1 (ActRII precursor). Therefore, structurally unordered amino acids adjacent to the core sequences bounded by these cysteines can truncate approximately 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, 27, 28, or 29 residues at the N-terminus and approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues at the C-terminus without necessarily altering ligand binding. Exemplary truncations of the ActRII extracellular domain include SEQ ID NOs: 2 and 3.

[0075] Therefore, the general formula of the active moiety of ActRII (e.g., ligand binding) is a polypeptide that contains, essentially consists of, or comprises amino acids 30-110 of SEQ ID NO: 1. Thus, an ActRII polypeptide starts at a residue corresponding to any one of amino acids 21-30 of SEQ ID NO: 1 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 1 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 It may contain, may essentially contain, or may be essentially contain, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRII (ending at any one of 22, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135). Other examples include positions selected from 21-30 of SEQ ID NO: 1 (e.g., starting with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), 22-30 (e.g., starting with any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), 23-30 (e.g., starting with any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30), and 24-30 (e.g., starting with any one of amino acids 24, 25, 26, 27, 28, 29, or 30). Starting from, and ending with any one of the amino acids 111-135 (e.g., amino acids 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 112-135 (e.g., amino acids 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,(ends with either 134 or 135), 113-135 (for example, amino acids 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), 120-135 (for example, amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 , ending with one of 131, 132, 133, 134, or 135), 130-135 (for example, ending with one of amino acids 130, 131, 132, 133, 134, or 135), 111-134 (for example, amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or (ends with one of 134), 111-133 (for example, amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133), 111-132 (for example, amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 Examples of constructs include those ending at any one of 1, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132, or 111-131 (for example, ending at any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, or 131). Variants within these ranges, in particular, are intended to include, essentially be, or consist of, amino acid sequences having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding portion of SEQ ID NO: 1. Therefore,In some embodiments, the ActRII polypeptide may, essentially be, or may be, a polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 1. If necessary, the ActRII polypeptide may include a polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 1 and includes 1, 2, 5, 10, or 15 or fewer conservative amino acid changes in the ligand-binding pocket. In some embodiments, the ActRII polypeptide is part of a homodimeric protein complex.

[0076] In certain embodiments, this disclosure relates to ActRII polypeptides (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or combination thereof), including fragments, functional variants, and modified forms thereof, and their use (e.g., to treat, prevent, or reduce pulmonary arterial hypertension). Preferably, the ActRII polypeptide is soluble (e.g., the extracellular domain of ActRII). In some embodiments, the ActRII polypeptide inhibits (e.g., Smad signaling) of one or more GDF / BMP ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15]. In some embodiments, the ActRII polypeptide binds to one or more GDF / BMP ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15]. In some embodiments, the ActRII polypeptide of this disclosure begins at a residue corresponding to amino acids 21-30 of SEQ ID NO: 1 (for example, starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 1 (for example, amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, It contains, essentially consists of, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRII (ending at any one of 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135).In some embodiments, the ActRII polypeptide contains, consists of, or is essentially composed of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 1. In certain embodiments, the ActRII polypeptide contains, consists of, or is essentially composed of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 21-135 of SEQ ID NO: 1. In some embodiments, the ActRII polypeptide comprises, consists of, or is essentially composed of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 23, 27, 30, and 41.

[0077] In some embodiments, the ActRII polypeptide contains, consists of, or is essentially composed of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some alternative embodiments, the ActRII polypeptide (e.g., SEQ ID NO: 23) may lack a C-terminal lysine. In some embodiments, the ActRII polypeptide lacking a C-terminal lysine is SEQ ID NO: 41. In some embodiments, the ActRII polypeptide contains, consists of, or is essentially derived from an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the patient is administered an ActRII polypeptide that contains, consists of, or is essentially derived from an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the patient is administered an ActRII polypeptide that contains, consists of, or is essentially derived from an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the patient is administered a combination of SEQ ID NO: 23 and SEQ ID NO: 41.

[0078] In certain embodiments, this disclosure relates to ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof). In some embodiments, the ActRII traps of this disclosure are variant ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) that include one or more mutations (e.g., addition, deletion, substitution, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRII polypeptide (e.g., “wild-type” or unmodified ActRII polypeptide) such that the variant ActRII polypeptide has one or more modified ligand-binding activities from the corresponding wild-type ActRII polypeptide. In preferred embodiments, the variant ActRII polypeptides of this disclosure retain at least one similar activity to the corresponding wild-type ActRII polypeptide. For example, preferred ActRII polypeptides bind to GDF11 and / or GDF8 and inhibit their function (e.g., antagonistize them). In some embodiments, the ActRII polypeptides of this disclosure further bind to and inhibit one or more ligands of GDF / BMP [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15]. Accordingly, this disclosure provides ActRII polypeptides having modified binding specificity to one or more ActRII ligands.

[0079] To illustrate, one or more mutations may be selected to increase the selectivity of the modified ligand-binding domains of GDF11 and / or GDF8 for one or more ActRII-binding ligands, particularly activin (activin A or activin B), such as activin A. If necessary, the modified ligand-binding domains may have a K+ ratio of at least 2, 5, 10, 20, 50, 100, or even 1000 times higher for activin binding compared to the wild-type ligand-binding domain. d K for GDF11 and / or GDF8 bond dIt has a ratio to . If necessary, the modified ligand-binding domain has an IC50 ratio of at least 2, 5, 10, 20, 50, 100 times, or even 1000 times higher IC50 ratio for activin inhibition compared to the wild-type ligand-binding domain. 50 IC regarding the inhibition of GDF11 and / or GDF8 50 It has a ratio to . If necessary, the modified ligand-binding domain has IC for activin inhibition. 50 ICs that are 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or even less than 1 / 1000 of that. 50 This inhibits GDF11 and / or GDF8.

[0080] In certain embodiments, the disclosure intends to induce specific mutations in ActRII polypeptides (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) to alter the glycosylation of the polypeptide. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally include asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid) in a tripeptide sequence that is specifically recognized by a suitable cellular glycosylation enzyme. The alteration may also be made by adding or substituting one or more serine or threonine residues into the polypeptide sequence (for O-linked glycosylation sites). Various amino acid substitutions or deletions at one or both of the first or third amino acid positions of the glycosylation recognition site (and / or amino acid deletions at the second position) result in nonglycosylation in the altered tripeptide sequence. Another means of increasing the number of carbohydrate moieties in a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the coupling method used, the sugars may be (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) amide groups of glutamine. The removal of one or more carbohydrate moieties present on the polypeptide may be achieved chemically and / or enzymatically. Chemical deglycosylation may involve, for example, exposure of the polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This process results in the cleavage of most or all sugars except linked sugars (N-acetylglucosamine or N-acetylgalactosamine) while keeping the amino acid sequence intact.Enzymatic cleavage of the carbohydrate moiety in polypeptides can be achieved by the use of various endoglycosidases and exoglycosidases, as described by Thotakura et al. [Meth. Enzymol. (1987) 138:350]. Since mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns that can be influenced by the amino acid sequence of the peptide, the polypeptide sequence may be adjusted as needed depending on the type of expression system used. Generally, polypeptides of this disclosure for use in humans may be expressed in mammalian cell lines that provide suitable glycosylation, such as HEK293 or CHO cell lines, but other mammalian expression cell lines are expected to be equally useful.

[0081] This disclosure further intends to describe methods for constructing sets of combinatorial mutants and truncation mutants of ActRII polypeptides (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or combination thereof). A pool of combinatorial mutants is particularly useful for identifying functionally active (e.g., GDF / BMP ligand-binding) ActRII sequences. The objective of screening such combinatorial libraries may be to construct polypeptide variants having altered properties, such as altered pharmacokinetics or altered ligand binding. Various screening assays are provided below, and such assays may be used to evaluate variants. For example, ActRII variants may be screened for their ability to bind to one or more GDF / BMP ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15], their ability to prevent the binding of GDF / BMP ligands to the ActRII polypeptide and its heteromultimers, and / or their ability to interfere with signaling induced by GDF / BMP ligands.

[0082] The activity of ActRII polypeptides (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) or their variants may also be tested in cell-based assays or in vivo assays. For example, the effect of ActRII polypeptides on the expression of genes involved in the pathogenesis of pulmonary arterial hypertension may be evaluated. This may be done in the presence of one or more recombinant ligand proteins [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15] as needed, and cells may be transfected to produce ActRII polypeptides and, if necessary, GDF / BMP ligands. Similarly, ActRII polypeptides may be administered to mice or other animals, and their effect on the pathogenesis of pulmonary arterial hypertension may be evaluated using methods recognized in the art. Likewise, the activity of ActRII polypeptides or their variants may be tested in hematopoietic progenitor cells for any effect on the growth of these cells, for example, by assays described herein and assays of general knowledge in the art. SMAD-responsive reporter genes may be used in such cell lines to monitor their effects on downstream signaling.

[0083] Combinatorial variants can be created that exhibit increased selectivity or generally increased potency compared to a reference ActRII polypeptide (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof). Such variants can be used in gene therapy protocols when expressed from recombinant DNA constructs. Similarly, mutagenesis can produce variants with dramatically different intracellular half-lives from the corresponding unmodified ActRII polypeptide. For example, the modified protein can be made either more stable or more unstable to proteolysis, or other cellular processes that would otherwise lead to the destruction or inactivation of the unmodified polypeptide. Such variants and the genes encoding them can be used to modulate polypeptide complex levels by modulating the polypeptide half-life. For example, a shorter half-life can produce more transient biological effects and, when part of an inducible expression system, may allow for tighter control of intracellular recombinant polypeptide complex levels. In Fc fusion proteins, mutations may be introduced in the linker (if any) and / or the Fc moiety to alter the ActRII polypeptide half-life.

[0084] The combinatorial library may be generated as a degenerate library of genes encoding a library of polypeptides, each containing at least a portion of the potential ActRII polypeptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated to the gene sequence so that the degenerate set of nucleotide sequences encoding the potential ActRII can be expressed as individual polypeptides, or instead as a larger set of fusion proteins (e.g., for phage display).

[0085] There are many methods for creating libraries of potential homologs from degenerate oligonucleotide sequences. The chemical synthesis of degenerate gene sequences can be performed in automated DNA synthesizers, and the synthesized genes can then be ligated into appropriate vectors for expression. The synthesis of degenerate oligonucleotides is well known in the art. [Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res. 11:477]. Such techniques have been used in the directional evolution of other proteins [Scott et al., (1990) Science 249:386-390; Roberts et al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and U.S. Patents 5,223,409, 5,198,346 and 5,096,815].

[0086] Alternatively, combinatorial libraries can be created using other forms of mutagenesis. For example, alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085], linker scanning mutagenesis [Gustin et al. (1993) By saturated mutagenesis [by Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science 232:316]; by PCR mutagenesis [by Meyers et al., (1986) Science 232:613]; by PCR mutagenesis [by Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by random mutagenesis including chemical mutagenesis [Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol Biol By screening using [7:32-34], for example, ActRII polypeptides of the present disclosure (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) can be prepared and isolated from the library.Linker scanning mutagenesis, particularly in combinatorial settings, is an attractive method for identifying truncated (bioactive) forms of ActRII polypeptides.

[0087] A wide range of techniques are known in the art for screening gene products of combinatorial libraries prepared by point mutation and truncation, and for screening cDNA libraries of gene products having certain properties. Such techniques would generally be adaptable for rapid screening of gene libraries prepared by combinatorial mutagenesis of ActRII polypeptides (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof). The most widely used techniques for screening large gene libraries typically involve cloning the gene library into a replicable expression vector, transforming suitable cells with the library of the resulting vector, and expressing the combinatorial gene under conditions where the detection of desired activity facilitates the relatively easy isolation of the vector encoding the gene from which the product was detected. Preferred assays include ligand [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15] binding assays and / or ligand-mediated cell signaling assays.

[0088] As will be recognized by those skilled in the art, most of the mutations, variants, or modifications described herein can be produced at the nucleic acid level, or in some cases, by post-translational modification or chemical synthesis. Such techniques are well known in the art, and some are described herein. In part, this disclosure identifies functionally active moieties (fragments) and variants of ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof) that can be used as guidance for producing and using other variant ActRII polypeptides within the scope of this disclosure provided herein.

[0089] In certain embodiments, functionally active fragments of the ActRII polypeptide of this disclosure can be obtained by screening polypeptides recombinantly produced from corresponding fragments of nucleic acids encoding the ActRII polypeptide. In addition, fragments can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid-phase f-Moc or t-Boc chemistry. The fragments can be generated (recombinantly or chemically synthesized) and tested to identify their peptidyl fragments that can function as antagonists (inhibitors) of the ActRII receptor and / or one or more ligands [e.g., GDF11, GDF8, activin A, activin B, GDF3, BMP4, BMP6, BMP10, and / or BMP15].

[0090] In certain embodiments, the ActRII polypeptides of this disclosure (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) may further include posttranslational modifications in addition to any naturally occurring posttranslational modifications in the ActRII polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the ActRII polypeptides may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the ligand-trap polypeptide may be tested as described herein for other ActRII variants. If the polypeptides of this disclosure are produced in cells by cleaving the nascent form of the polypeptide, posttranslational processing may also be important for the correct folding and / or function of the protein. Different cell types (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) may be selected to ensure precise modification and processing of ActRII polypeptides, as they possess specific cellular mechanisms and characteristic processes for such post-translational activity.

[0091] In certain embodiments, the ActRII polypeptides of this disclosure (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) include fusion proteins having at least a portion (domain) of an ActRII polypeptide and one or more heterologous portions (domains). Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP), or human serum albumin. The fusion domains may be selected to confer desired properties. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as a resin conjugated with glutathione, amylase, and nickel or cobalt, are used. Many such matrices are available in “kit” form, such as the QIAexpress™ system (Qiagen), which is useful with the Pharmacia GST purification system and (HIS6) fusion partner. Another example is that fusion domains may be selected to facilitate the detection of ActRII polypeptides. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and “epitope tags,” which are usually short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein from them. The released protein can then be isolated from the fusion domain by subsequent chromatographic separation.Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains, and functional domains (which confer additional biological function) including, for example, a constant domain derived from immunoglobulin (e.g., an Fc domain).

[0092] In certain embodiments, the ActRII polypeptides of the present disclosure (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) include one or more modifications that can “stabilize” the polypeptide. “Stabilizing” means any modification that increases the in vitro half-life, serum half-life, whether this is due to reduced drug breakdown, reduced renal clearance, or other pharmacokinetic effects. For example, such modifications enhance the shelf life of the polypeptide, enhance the circulating half-life of the polypeptide, and / or reduce the proteolysis of the polypeptide. Such stabilizing modifications include, but are not limited to, fusion proteins (e.g., fusion proteins comprising an ActRII polypeptide domain and a stabilizer domain), modifications of glycosylation sites (e.g., adding a glycosylation site to the polypeptide of the present disclosure), and modifications of carbohydrate moieties (e.g., removing a carbohydrate moiety from the polypeptide of the present disclosure). As used herein, the term “stabilizer domain” refers not only to a fusion domain (e.g., an immunoglobulin Fc domain) as in the case of a fusion protein, but also to non-proteinoid modifications such as a carbohydrate moiety, or non-proteinoid moieties such as polyethylene glycol. In certain preferred embodiments, the ActRII polypeptide is fused with a heterogeneous domain that stabilizes the polypeptide (a “stabilizer” domain), preferably a heterogeneous domain that increases the in vivo stability of the polypeptide. Fusion with a constant domain of immunoglobulin (e.g., an Fc domain) is known to confer desirable pharmacokinetic properties to a wide range of proteins. Similarly, fusion with human serum albumin can confer desirable properties.

[0093] An example of a native amino acid sequence that may be used for the Fc portion (G1Fc) of human IgG1 is shown below (SEQ ID NO: 11). Dotted underlines indicate hinge regions, and solid underlines indicate positions with naturally occurring variants. In part, this disclosure provides polypeptides containing, essentially derived from, or comprising amino acid sequences having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11. Naturally occurring variants of G1Fc would include E134D and M136L, according to the numbering system used in SEQ ID NO: 11 (see Uniprot P01857). [ka]

[0094] If necessary, the IgG1 Fc domain may have one or more mutations in residues such as Asp-265, lysine 322, and Asn-434. In certain cases, mutant IgG1 Fc domains with one or more of these mutations (e.g., the Asp-265 mutation) have reduced binding ability to Fcγ receptors compared to wild-type Fc domains. In other cases, mutant Fc domains with one or more of these mutations (e.g., the Asn-434 mutation) have increased binding ability to MHC class I-associated Fc receptors (FcRNs) compared to wild-type IgG1 Fc domains.

[0095] An example of a native amino acid sequence that may be used for the Fc portion (G2Fc) of human IgG2 is shown below (SEQ ID NO: 12). Dotted underlines indicate hinge regions, and double underlines indicate locations in the sequence where database discrepancies exist (according to UniProt P01859). In part, this disclosure provides polypeptides comprising, essentially derived from, or consisting of amino acid sequences having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12. [ka]

[0096] Two examples of amino acid sequences that may be used for the Fc portion (G3Fc) of human IgG3 are shown below. The hinge region in G3Fc can be up to four times longer than in other Fc chains and contains three identical 15-residue segments preceded by a similar 17-residue segment. The first G3Fc sequence shown below (SEQ ID NO: 13) contains a short hinge region consisting of a single 15-residue segment, while the second G3Fc sequence (SEQ ID NO: 14) contains a full-length hinge region. In each case, the dotted underline indicates the hinge region, and the solid underline indicates the position with a naturally occurring variant according to UniProt P01859. In part, the present disclosure provides polypeptides comprising, essentially, or consisting of amino acid sequences having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 13 and 14. [ka]

[0097] Naturally occurring variants in G3Fc (see, e.g., Uniprot P01860) include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del, and F221Y when converted to the numbering system used in Sequence ID No. 13, and this disclosure provides fusion proteins containing a G3Fc domain containing one or more of these variant forms. In addition, the human immunoglobulin IgG3 gene (IGHG3) exhibits structural polymorphisms characterized by different hinge lengths [see Uniprot P01859]. Specifically, variant WIS lacks most of the V region and all of the CH1 region. It has an extra interchain disulfide bond at position 7 in addition to the normally present position 11 in the hinge region. Variant ZUC lacks most of the V region, all of the CH1 region, and part of the hinge. The variant OMM may represent an allele or another gamma chain subclass. This disclosure provides further fusion proteins containing a G3Fc domain containing one or more of these variants.

[0098] An example of a native amino acid sequence that may be used for the Fc portion (G4Fc) of human IgG4 is shown below (SEQ ID NO: 15). The dotted underline indicates the hinge region. In part, the present disclosure provides polypeptides comprising, essentially, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15. [ka]

[0099] Various engineered mutations in the Fc domain are presented herein with respect to the G1Fc sequence (SEQ ID NO: 11), and similar mutations in G2Fc, G3Fc, and G4Fc can be derived from their alignment with G1Fc in FIG. 4. Due to the unequal hinge lengths, similar Fc positions based on the isotype alignment (FIG. 4) have different amino acid numbers in SEQ ID NOs: 11, 12, 13, 14, and 15. It can also be understood that a given amino acid position in an immunoglobulin sequence consisting of the hinge, C H 1, hinge, C H 2 and C H 3 regions (when encompassing the entire IgG1 heavy chain constant domain) (such as SEQ ID NOs: 11, 12, 13, 14, and 15) is specified by a different number than the same position. For example, the correspondence between the human G1Fc sequence (SEQ ID NO: 11), the human IgG1 heavy chain constant domain (Uniprot P01857), and selected C H 3 positions in the human IgG1 heavy chain is as follows.

[0100]

Table A

[0101] ​​​​Various methods are known in the art for increasing the desired pairing of Fc-containing fusion polypeptide chains in single-cell systems to produce preferred asymmetric fusion proteins in acceptable yields [Klein et al (2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. Methods for obtaining desired pairing of Fc-containing chains include, but are not limited to, charge-based pairing (electrostatic steering), "knob-into-hole" stereopairing, seedbody pairing, and leucine zipper-based pairing [Ridgway et al (1996) Protein Eng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al (2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010); 285:19637-19646; Wranik et al (2012) J Biol Chem [287:43331-43339; US Patent No. 5932448; WO1993 / 011162; WO2009 / 089004 and WO2011 / 034605].

[0102] It is understood that the different elements of a fusion protein (e.g., an immunoglobulin Fc fusion protein) can be arranged in any manner that matches the desired functionality. For example, the ActRII polypeptide domain may be placed C-terminus relative to the heterologous domain, or the heterologous domain may be placed C-terminus relative to the ActRII polypeptide domain. The ActRII polypeptide domain and the heterologous domain do not need to be adjacent in the fusion protein, and additional domains or amino acid sequences may be included C-terminus or N-terminus of either domain, or between the domains.

[0103] For example, an ActRII receptor fusion protein may contain the amino acid sequence shown in formula ABC. The B portion corresponds to the ActRII polypeptide domain (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof). The A and C portions may independently consist of 0, 1, or more than 1 amino acids, and both A and C portions, if present, are heterogeneous to B. The A and / or C portions may be bound to the B portion via a linker sequence. The linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, or 2-3 glycine residues) or glycine and proline residues, and may contain, for example, a single sequence of threonine / serine and glycine, or a repeat sequence of threonine / serine and / or glycine, such as a singlet or repeat of GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), TGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), TGGG (SEQ ID NO: 20), SGGG (SEQ ID NO: 21), or GGGGS (SEQ ID NO: 22). In certain embodiments, the ActRII fusion protein comprises the amino acid sequence shown in formula ABC, where A is a leader (signal) sequence, B consists of the ActRII polypeptide domain, and C is a polypeptide moiety that enhances one or more of the following: in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. In certain embodiments, the ActRII fusion protein comprises the amino acid sequence shown in formula ABC, where A is the TPA reader sequence, B is the ActRII receptor polypeptide domain, and C is the immunoglobulin Fc domain. A preferred fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 23, 27, 30, and 41.

[0104] In preferred embodiments, the ActRII polypeptide used according to the methods described herein is an isolated polypeptide. As used herein, an isolated protein or polypeptide is one that has been isolated from its natural environment. In some embodiments, the polypeptides of this disclosure are purified to a purity of 95%, 96%, 97%, 98%, or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). Methods for evaluating purity are well known in the art [see, for example, Flatman et al., (2007) J. Chromatogr. B 848:79-87]. In some embodiments, the ActRII polypeptide used according to the methods described herein is a recombinant polypeptide.

[0105] The ActRII polypeptides of this disclosure can be produced by various techniques known in the art. For example, the polypeptides of this disclosure can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, the polypeptides of this disclosure, including their fragments or variants, may be recombinantly produced using various expression systems [e.g., E. coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus], as is well known in the art. In further embodiments, modified or unmodified polypeptides of the present disclosure may be produced by digestion of recombinantly produced full-length ActRII polypeptides, for example, by using proteases such as trypsin, thermolysin, chymotrypsin, pepsin, or pair-forming basic amino acid converting enzymes (PACE). Protein cleavage sites can be identified using computer analysis (using commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.). Alternatively, such polypeptides may be produced from recombinantly produced full-length ActRII polypeptides using chemical cleavage (e.g., cyanogen bromide, hydroxylamine, etc.).

[0106] 3. Nucleic acid encoding ActRII polypeptide In certain embodiments, the disclosure provides isolated nucleic acids and / or recombinant nucleic acids encoding ActRII polypeptides (e.g., ActRIIA polypeptides, ActRIIB polypeptides, or combinations thereof), including fragments, functional variants, and fusion proteins.

[0107] As used herein, isolated nucleic acids refer to nucleic acid molecules separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0108] In certain embodiments, it is understood that the nucleic acid encoding the ActRII polypeptide of the present disclosure comprises a nucleic acid that is any one variant of SEQ ID NO: 4, 5, or 28. The variant nucleotide sequence comprises a sequence that differs due to the substitution, addition, or deletion of one or more nucleotides, including the allele variant, and therefore comprises a coding sequence that is different from the nucleotide sequence specified in any one of SEQ ID NO: 4, 5, or 28.

[0109] In certain embodiments, the ActRII polypeptides of the Disclosure are encoded by isolated nucleic acid sequences and / or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 4, 5, or 28. Those skilled in the art will understand that nucleic acid sequences and variants thereof that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequences complementary to SEQ ID NOs: 4, 5, or 28 are also within the scope of the Disclosure. In further embodiments, the nucleic acid sequences of the Disclosure may be isolated, recombinant, and / or fused with heterologous nucleotide sequences, or present in a DNA library.

[0110] In other embodiments, the nucleic acids of the Disclosure also include nucleotide sequences that hybridize under highly stringent conditions with a nucleotide sequence specified in SEQ ID NO: 4, 5, or 28, a complementary sequence of SEQ ID NO: 4, 5, or 28, or a fragment thereof. As discussed above, those skilled in the art will readily understand that the appropriate stringency conditions that promote DNA hybridization can be varied. For example, hybridization can be performed with 6.0 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing with 2.0 × SSC at 50°C. For example, the salt concentration in the washing step can be selected from low stringency of about 2.0 × SSC at 50°C to high stringency of about 0.2 × SSC at 50°C. In addition, the temperature in the washing step can be increased from low stringency conditions of about 22°C (room temperature) to high stringency conditions of about 65°C. Both temperature and salt concentration may be varied, or the temperature or salt concentration may be kept constant while other variables are varied. In one embodiment, the disclosure provides nucleic acids that hybridize under low stringency conditions of washing with 6×SSC at room temperature followed by 2×SSC at room temperature.

[0111] Isolated nucleic acids that differ from those shown in Sequence ID No. 4, 5, or 28 in terms of degeneracy in the genetic code are also within the scope of this disclosure. For example, some amino acids are designated by one or more triplets. Codons or synonyms that identify the same amino acid (e.g., CAU and CAC are synonyms for histidine) may result in “silent” mutations that do not affect the amino acid sequence of the protein. However, it is expected that in mammalian cells there will be DNA sequence polymorphisms that result in changes in the amino acid sequence of the protein in question. Those skilled in the art will understand that these variant forms in one or more nucleotides (up to about 3-5% of nucleotides) of the nucleic acid encoding a particular protein may exist among individuals of a given species due to naturally occurring allelic variant forms. All such nucleotide variant forms and the resulting amino acid polymorphisms are within the scope of this disclosure.

[0112] In certain embodiments, the recombinant nucleic acids of this disclosure may be operably ligated to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art and can be used in various host cells. Typically, one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are intended by this disclosure. The promoter may be a naturally occurring promoter or a hybrid promoter combining elements of one or more promoters. The expression construct may be present in the cell on an episome such as a plasmid, or the expression construct may be inserted into a chromosome. In some embodiments, the expression vector contains a selection marker gene to allow selection of transformed host cells. Selection marker genes are well known in the art and can be modified depending on the host cell used.

[0113] In certain embodiments, the nucleic acids disclosed herein are provided in expression vectors comprising a nucleotide sequence encoding an ActRII polypeptide (e.g., ActRIIA polypeptide, ActRIIB polypeptide, or a combination thereof) operably ligated to at least one regulatory sequence. The regulatory sequence is recognized in the art and selected to direct the expression of the ActRII polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of the wide variety of expression regulatory sequences that control the expression of a DNA sequence when operably ligated may be used in these vectors to express a DNA sequence encoding an ActRII polypeptide. Such useful expression regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the pre-early promoters of adenoviruses or cytomegaloviruses, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the regulatory regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycoseptic enzymes, promoters of acid phosphatases, such as Pho5, the yeast α-conjugation factor promoter, the polyhedron promoter of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells, or their viruses, as well as various combinations thereof. It should be understood that the design of expression vectors may depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. The copy number of the vector, its ability to control copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0114] The recombinant nucleic acids of this disclosure can be produced by ligating a cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vehicles for the production of recombinant ActRII polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids for expression in prokaryotic cells such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0115] Some mammalian expression vectors contain both prokaryotic sequences to promote vector reproduction in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for eukaryotic cell transfection. Some of these vectors are modified with bacterial plasmid-derived sequences, such as pBR322, to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP, and p205) can be used for transient protein expression in eukaryotic cells. Examples of other viral (including retrovirus) expression systems can be found below in the description of gene therapy delivery systems. Various methods used in plasmid preparation and host organism transformation are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, and for general recombination procedures, see, for example, Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant polypeptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., β-gal-containing pBlueBac III).

[0116] In preferred embodiments, vectors such as Pcmv-Script vectors (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.), and pCI-neo vectors (Promega, Madison, Wisc.) are designed for the production of the target ActRII polypeptide in CHO cells. As is evident, the target gene construct can be used to induce the expression of the target ActRII polypeptide in cells grown in culture, for example, to produce a protein containing a fusion protein or a variant protein, for purification purposes.

[0117] This disclosure also relates to host cells transfected with recombinant genes containing one or more coding sequences of the ActRII polypeptide in question. The host cells may be any prokaryotic or eukaryotic cells. For example, the ActRII polypeptides of this disclosure may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells [e.g., Chinese hamster ovary (CHO) cell line]. Other suitable host cells are known to those skilled in the art.

[0118] Accordingly, this disclosure further relates to methods for generating the ActRII polypeptide of interest. For example, host cells transfected with an expression vector encoding the ActRII polypeptide can be cultured under suitable conditions capable of inducing the expression of the ActRII polypeptide. The polypeptide may be secreted and isolated from a mixture of cells and culture medium containing the polypeptide. Alternatively, the ActRII polypeptide may be retained in the cytoplasm, or may be recovered and lysed in membrane fractions and cells, as well as in isolated proteins. The cell culture comprises host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art. The polypeptide of interest can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using antibodies specific to a particular epitope of the ActRII polypeptide, and affinity purification using a drug that binds to the domain fused to the ActRII polypeptide (for example, the ActRII-Fc fusion protein can be purified using a protein A column). In some embodiments, the ActRII polypeptide is a fusion protein containing a domain that facilitates its purification.

[0119] In some embodiments, purification is achieved by a series of column chromatography steps, for example, comprising three or more of the following in any order: protein A chromatography, Q Sepharose chromatography, phenyl Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be completed by viral filtration and buffer exchange. ActRII proteins may be purified to purities of >90%, >95%, >96%, >98%, or >99% as determined by size exclusion chromatography, and to purities of >90%, >95%, >96%, >98%, or >99% as determined by SDS PAGE. The target level of purity must be sufficient to achieve the desired results in mammalian systems, particularly in non-human primates, rodents (mice), and humans.

[0120] In another embodiment, a fusion gene encoding a purified leader sequence, for example, a poly-(His) / enterokinase cleavage site sequence at the N-terminus of a desired portion of a recombinant ActRII polypeptide, is Ni 2+ Affinity chromatography using a metal resin may enable the purification of the expressed fusion protein. The purified leader sequence can then be removed by subsequent treatment with enterokinase to provide a purified ActRII polypeptide. See, for example, Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al. (1991) PNAS USA 88:8972.

[0121] Techniques for constructing fusion genes are well known. Essentially, the conjugation of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques using blunt or twisted ends for ligation, restriction enzyme digestion to provide suitable ends, sticky end filling where appropriate, alkaline phosphatase treatment to avoid undesirable conjugations, and enzymatic ligation. In another embodiment, fusion genes can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that create complementary overhangs between two consecutive gene fragments, which can then be annealed to generate a chimeric gene sequence. See, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992.

[0122] 4. How to use In part, this disclosure relates to a method for treating pulmonary arterial hypertension (PAH), comprising the step of administering an effective amount of the ActRII polypeptide described herein to a patient in need. In some embodiments, this disclosure envisions a method for treating, preventing, or reducing the rate of progression and / or severity of one or more complications of pulmonary arterial hypertension, comprising the step of administering an effective amount of the ActRII polypeptide described herein to a patient in need. In some embodiments, the ActRII polypeptide is administered in a dosage range of 0.1 mg / kg to 2.0 mg / kg (e.g., 0.3 mg / kg or 0.7 mg / kg). In some embodiments, administration of ActRII polypeptide results in changes in one or more hemodynamic or functional parameters (e.g., reduction in pulmonary vascular resistance (PVR); increase in 6-minute walk distance (6MWD); decrease in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; prevention or delay of progression of the World Health Organization (WHO) recognized class of pulmonary hypertension; promotion or increase of regression of the WHO recognized class of pulmonary hypertension; improvement in right ventricular function; and improvement in pulmonary artery pressure).

[0123] These methods are particularly intended for the therapeutic and prophylactic treatment of animals, more specifically humans. The terms “subject,” “individual,” or “patient” are interchangeable throughout this specification and refer to either human or non-human animals. These terms include mammals, e.g., humans, non-human primates, laboratory animals, domesticated animals (including cattle, pigs, camels, etc.), companion animals (e.g., dogs, cats, other domesticated animals, etc.), and rodents (e.g., mice and rats). In certain embodiments, the patient, subject, or individual is human.

[0124] The terms “treatment,” “treating,” and “relieving” are used herein to generally mean obtaining a desired pharmacological and / or physiological effect, and may also be used to mean improving, reducing, and / or decreasing the severity of one or more clinical complications of the treated condition (e.g., PAH). The effect may be preventive in that it completely or partially delays the onset or recurrence of the disease, condition, or its complications, and / or therapeutic in that it partially or completely cures the disease or condition and / or adverse effects resulting from the disease or condition. “Treatment,” as used herein, encompasses any treatment of a disease or condition in a mammal, particularly a human. As used herein, a “preventive” therapeutic agent of a disorder or condition means a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset of the disease or condition compared to an untreated control sample.

[0125] Generally, the treatment or prevention of the diseases or conditions described in this disclosure (e.g., PAH) is achieved by administering one or more ActRII polypeptides of this disclosure in an “effective dose.” The effective dose of a drug refers to the amount effective to achieve the desired therapeutic or preventive outcome over the required dosage and duration. The “therapeutic effective dose” of a drug in this disclosure may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the drug’s ability to induce the desired response in the individual. The “preventive effective dose” refers to the amount effective to achieve the desired preventive outcome over the required dosage and duration.

[0126] In certain embodiments, the Disclosure intends to describe the use of ActRII polypeptides in combination with one or more additional activators or other supportive therapies to treat or prevent a disease or condition (e.g., PAH). As used herein, “in combination with,” “combined with,” or “joint” administration refers to any form of administration in which the additional activator or supportive therapy (e.g., second, third, fourth, etc.) remains effective in the body (e.g., multiple compounds are effective simultaneously in the patient for a period of time, which may include a synergistic effect of these compounds). Efficacy does not have to correlate with a measurable concentration of the drug in blood, serum, or plasma. For example, different therapeutic compounds may be administered in the same formulation or in separate formulations, concurrently or sequentially, on different schedules. Thus, subjects receiving such treatment may benefit from the combined effects of different activators or therapies. One or more ActRII polypeptides of the Disclosure may be administered concurrently, before, or after one or more other additional agents or supportive therapies, e.g., those disclosed herein. Generally, each activator or therapy is administered in a dose and / or time schedule determined for the specific drug. The specific combination used in the regimen takes into account the compatibility of the ActRII polypeptides of this disclosure with additional activators or supportive therapies, and / or the desired effect.

[0127] Overview of the WHO classification The pulmonary arterial hypertension conditions treated by the methods described herein may include one or more of the conditions recognized in accordance with the World Health Organization (WHO). See, for example, Simonneau (2019) Eur Respir J: 53:1801913.

[0128] [Table 1]

[0129] The clinical purpose of PAH classification is to categorize the clinical conditions associated with PAH into specific subgroups according to their pathophysiological mechanisms, clinical presentation, hemodynamic characteristics, and treatment strategies. This clinical classification may be updated as new data become available on the above characteristics or when additional clinical entities are considered.

[0130] As used herein, the term “pulmonary hemodynamic parameters” refers to any parameters used to describe or assess blood flow through the cardiac and pulmonary vascular systems. Examples of pulmonary hemodynamic parameters include, but are not limited to, mean pulmonary artery pressure (mPAP), diastolic pulmonary artery pressure (dPAP) [also known as pulmonary artery diastolic pressure (PADP)], systolic pulmonary artery pressure (sPAP) [also known as pulmonary artery systolic pressure (PASP)], mean right atrial pressure (mRAP), pulmonary capillary wedge pressure (PCWP) [also known as pulmonary artery wedge pressure (PAWP)], pulmonary vascular resistance (PVR), and cardiac output (CO).

[0131] Many of the pulmonary hemodynamic parameters listed above are interrelated. For example, PVR is related to mPAP, PCWP, and CO according to the following equation: PVR = (mPAP - PCWP) / CO [in Woods units] PVR measures the resistance to flow imposed by the pulmonary vascular system without the influence of left-sided filling pressure. PVR is calculated using the following formula: PVR = TPG × 80 / CO[Unit: dyne-second-cm] -5 ] or PVR = (mPAP - PCWP) × 80 / CO [Unit: dyne-second-cm] -5 ] It can also be measured according to this method.

[0132] In some embodiments, total peripheral resistance (TPR) can be measured using the following equation: TPR=mPAP / CO

[0133] According to some embodiments, the contribution of the precapillary pulmonary artery to PH may be reflected by an elevated PVR. In some embodiments, a normal PVR is 20–130 dyne-seconds–cm². -5 Or it is 0.5 to 1.1 wood units. According to some embodiments, an increased PVR may refer to a PVR greater than 2 wood units, greater than 2.5 wood units, greater than 3 wood units, or greater than 3.5 wood units.

[0134] As yet another example, mPAP is related to dPAP and sPAP according to the following equation: mPAP = (2 / 3)dPAP + (1 / 3)sPAP

[0135] Furthermore, dPAP and sPAP can be used to calculate pulse pressure (mmHg) using the following formula: pulse pressure = sPAP - dPAP.

[0136] Pulse pressure is calculated using the following formula: Pulmonary artery compliance (mI.mmHg) -1 ) = stroke volume / pulse pressure can be used to calculate pulmonary artery compliance.

[0137] In some embodiments, pulmonary hemodynamic parameters are measured directly, such as during right heart catheterization. In other embodiments, pulmonary hemodynamic parameters are estimated and / or evaluated by other techniques such as magnetic resonance imaging (MRI) or echocardiography.

[0138] Exemplary pulmonary hemodynamic parameters include mPAP, PAWP, and PVR. One or more pulmonary hemodynamic parameters can be measured by any appropriate procedure, such as by right heart catheterization or echocardiography. Table 2 shows the various hemodynamic characteristics of PH and PAH.

[0139] [Table 2]

[0140] The clinical classifications or hemodynamic characteristics of PAH described herein, and related diagnostic parameters, may be updated or changed based on the availability of new or existing data sources, or when additional clinical entities are considered.

[0141] Characteristics of PAH Pulmonary arterial hypertension (PH, Group 1 according to the WHO) is a severe, progressive, and life-threatening disease of the pulmonary vascular system characterized by severe vasoconstriction and abnormal proliferation of smooth muscle cells in the walls of the pulmonary arteries. Severe constriction of the pulmonary blood vessels results in extremely high pulmonary artery pressure. These high pressures make it difficult for the heart to pump blood through the lungs to supply oxygen. Patients with PAH suffer from extreme shortness of breath as the heart tries to pump blood against these high pressures. Patients with PAH typically develop a marked increase in PVR and a persistent increase in mPAP, which ultimately leads to right ventricular failure and death. Patients diagnosed with PAH have a poor prognosis, as well as a impaired quality of life, with an average life expectancy of 2 to 5 years from the time of diagnosis if left untreated.

[0142] Various factors contribute to the pathogenesis of pulmonary hypertension, including the proliferation of lung cells (i.e., hyperplasia) that can contribute to vascular remodeling. For example, pulmonary vascular remodeling is primarily caused by the proliferation of arterial endothelial cells and smooth muscle cells in patients with pulmonary hypertension. Overexpression of various cytokines is thought to promote pulmonary hypertension. Furthermore, it has been found that pulmonary hypertension can arise from the overproliferation of pulmonary artery smooth cells and pulmonary endothelial cells. Moreover, progressive PAH can be characterized by muscularization of distal pulmonary arterioles, concentric endocardium thickening, and occlusion of vascular lumen due to endothelial cell proliferation. Pietra et al., J. Am. Coll. Cardiol., 43:255-325 (2004).

[0143] PAH can be diagnosed based on mean pulmonary artery pressure above 25 mmHg at rest (or above 20 mmHg under updated guidelines) and normal pulmonary artery capillary wedge pressure. PAH can result in shortness of breath, dizziness, syncope, and other symptoms, all of which worsen with exercise. PAH can be a severe disorder accompanied by significantly reduced exercise tolerance and heart failure. There are two main types of PAH: idiopathic PAH (e.g., PAH with no identified predisposition) and hereditary PAH (e.g., PAH associated with mutations in BMPR2, ALK1, ENG, SMAD9, CAV1, KCNK3, or EIF2AK4). In 70% of cases of familial PAH, the mutation is located in the BMPR2 gene. Risk factors for the development of PAH include a family history of PAH, drug and toxin use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital cardiac anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangioma, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus erythematosus). PAH may be associated with calcium channel blockers, the apparent venous / capillary (PVOD / PCH) involvement, and long-term responders to persistent PH in neonatal syndromes.

[0144] Diagnosis of PAH The diagnosis of PAH, including functional groups, can be determined based on symptoms and health examination, using a comprehensive review of a set of parameters to determine whether hemodynamic and other criteria are met. Some of the criteria that may be considered include the patient's clinical presentation (e.g., shortness of breath, fatigue, weakness, anguina, fainting, dry cough, exercise-induced nausea, and vomiting), electrocardiogram (ECG) results, chest X-ray results, pulmonary function tests, arterial blood gases, echocardiogram results, ventilation / perfusion lung scan results, high-resolution computed tomography results, contrast-enhanced computed tomography results, pulmonary angiography results, magnetic resonance imaging, blood tests (e.g., biomarkers such as BNP or NT-proBNP), immunology, abdominal ultrasound scan, right heart catheterization (RHC), vascular reactivity, and genetic testing. See, for example, Galie N., et al Euro Heart J. (2016) 37, 67-119.

[0145] In some embodiments, biomarkers can be used to aid in the diagnosis of PAH. For example, in some embodiments, the biomarker is a marker of vascular dysfunction (e.g., asymmetric dimethylarginine (ADMA), endothelin-1, angiopoietin, or von Willebrand factor). In some embodiments, the biomarker is a marker of inflammation (C-reactive protein, interleukin-6, chemokines). In some embodiments, the biomarker is a marker of myocardial stress [e.g., atrial natriuretic peptide, brain natriuretic peptide (BNP) / NT-proBNP, or troponin]. In some embodiments, the biomarker is a marker of low CO and / or tissue hypoxia [e.g., pCO2, uric acid, growth and differentiation factor 15 (GDF15), or osteopontin]. In some embodiments, the biomarker is a marker of secondary organ injury [e.g., creatinine or bilirubin]. See, for example, Galie N., et al Euro Heart J. (2016) 37, 67-119.

[0146] pH measurement In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of pulmonary arterial hypertension (PAH), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need. In some embodiments, the method relates to treating a PAH patient having idiopathic PAH. In some embodiments, the method relates to treating a PAH patient having hereditary PAH (e.g., PAH resulting from one or more mutations in BMPR2, ALK-1, ENG, SMAD9, CAV1, and KCNK3). In some embodiments, the method relates to treating a PAH patient having hereditary PAH resulting from an unknown mutation. In some embodiments, the method relates to treating a PAH patient having drug or toxin-induced PAH. In some embodiments, the method relates to treating a PAH patient having PAH associated with connective tissue disease. In some embodiments, the method relates to treating a PAH patient having PAH associated with HIV infection. In some embodiments, the method relates to treating PAH patients with PAH associated with portal hypertension. In some embodiments, the method relates to treating PAH patients with PAH associated with schistosomiasis. In some embodiments, the method relates to treating PAH patients classified as long-term responders to calcium channel blockers. In some embodiments, the method relates to treating PAH patients with evident venous / capillary (PVOD / PCH) involvement. In some embodiments, the method relates to treating PAH patients with persistent pulmonary hypertension (PH) of neonatal syndrome. In some embodiments, the method relates to treating PAH patients with PAH associated with a simple, congenital systemic-to-pulmonary shunt at least one year after shunt repair.

[0147] mPAP In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of pulmonary arterial hypertension (PAH), comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), wherein the patient has a resting mean pulmonary artery pressure (mPAP) of at least 20 mmHg (e.g., 20, 25, 30, 35, 40, 45, or 50 mmHg). In some embodiments, the method relates to a patient having a resting mPAP of at least 20 mmHg. In some embodiments, the method relates to a patient having a resting mPAP of at least 25 mmHg. In some embodiments, the method relates to a patient having a resting mPAP of at least 30 mmHg. In some embodiments, the method relates to a patient having a resting mPAP of at least 35 mmHg. In some embodiments, the method relates to a patient having a resting mPAP of at least 40 mmHg. In some embodiments, the method relates to a patient having a resting mPAP of at least 45 mmHg. In some embodiments, the method relates to a patient having a resting mPAP of at least 50 mmHg.

[0148] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving pulmonary artery pressure in the patient. In some embodiments, improvement of pulmonary artery pressure is a reduction in mean pulmonary artery pressure (mPAP). In some embodiments, the method relates to reducing mPAP. In some embodiments, the method relates to reducing the patient's mPAP by at least 1 mmHg. In some embodiments, the method relates to reducing the patient's mPAP by at least 2 mmHg. In some embodiments, the method relates to reducing the patient's mPAP by at least 3 mmHg. In certain embodiments, the method relates to reducing the patient's mPAP by at least 5 mmHg. In certain embodiments, the method relates to reducing the patient's mPAP by at least 7 mmHg. In certain embodiments, the method relates to reducing a patient's mPAP by at least 10 mmHg. In certain embodiments, the method relates to reducing a patient's mPAP by at least 12 mmHg. In certain embodiments, the method relates to reducing a patient's mPAP by at least 15 mmHg. In certain embodiments, the method relates to reducing a patient's mPAP by at least 20 mmHg. In certain embodiments, the method relates to reducing a patient's mPAP by at least 25 mmHg.

[0149] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's mPAP by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to reducing the patient's mPAP by at least 1%. In some embodiments, the method relates to reducing the patient's mPAP by at least 5%. In some embodiments, the method relates to reducing the patient's mPAP by at least 10%. In some embodiments, the method relates to reducing the patient's mPAP by at least 15%. In some embodiments, the method relates to reducing the patient's mPAP by at least 20%. In some embodiments, the method relates to reducing the patient's mPAP by at least 25%. In some embodiments, the method relates to reducing the patient's mPAP by at least 30%. In some embodiments, the method relates to reducing the patient's mPAP by at least 35%. In some embodiments, the method relates to reducing the patient's mPAP by at least 40%. In some embodiments, the method relates to reducing the patient's mPAP by at least 45%. In some embodiments, the method relates to reducing the patient's mPAP by at least 50%. In some embodiments, the method relates to reducing the patient's mPAP by at least 55%. In some embodiments, the method relates to reducing the patient's mPAP by at least 60%. In some embodiments, the method relates to reducing the patient's mPAP by at least 65%. In some embodiments, the method relates to reducing the patient's mPAP by at least 70%. In some embodiments, the method relates to reducing the patient's mPAP by at least 75%.In some embodiments, the method relates to reducing the patient's mPAP by at least 80%. In some embodiments, the method relates to reducing the patient's mPAP by at least 85%. In some embodiments, the method relates to reducing the patient's mPAP by at least 90%. In some embodiments, the method relates to reducing the patient's mPAP by at least 95%. In some embodiments, the method relates to reducing the patient's mPAP by at least 100%.

[0150] mRAP As PAH progresses, increased pulmonary vascular resistance to blood flow leads to increased right atrial pressure (RAP) and right heart failure. Patients with right heart failure typically have an increased ratio of right atrial pressure (RAP) to pulmonary artery wedge pressure (PAWP). In certain embodiments, the present disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of pulmonary arterial hypertension (PAH), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has a mean resting right atrial pressure (mRAP) of at least 5 mmHg (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20, 21, 22, 23, 24, or 25 mmHg). In some embodiments, the method relates to a patient having a resting mRAP of at least 5 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 6 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 7 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 8 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 9 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 10 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 11 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 12 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 13 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 14 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 15 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 16 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 17 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 18 mmHg.In some embodiments, the method relates to a patient having a resting mRAP of at least 19 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 20 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 21 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 22 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 23 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 24 mmHg. In some embodiments, the method relates to a patient having a resting mRAP of at least 25 mmHg.

[0151] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving mean right atrial pressure in the patient. In some embodiments, improvement of mean right atrial pressure (mRAP) is a reduction of mRAP. In some embodiments, the method relates to reducing mRAP. In some embodiments, the method relates to reducing the patient's mRAP by at least 1 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 2 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 3 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 4 mmHg. In certain embodiments, the method relates to reducing the patient's mRAP by at least 5 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 6 mmHg. In certain embodiments, the method relates to reducing the patient's mRAP by at least 7 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 8 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 9 mmHg. In certain embodiments, the method relates to reducing the patient's mRAP by at least 10 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 11 mmHg. In certain embodiments, the method relates to reducing the patient's mRAP by at least 12 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 13 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 14 mmHg. In certain embodiments, the method relates to reducing the patient's mRAP by at least 15 mmHg.In some embodiments, the method relates to reducing the patient's mRAP by at least 16 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 17 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 18 mmHg. In some embodiments, the method relates to reducing the patient's mRAP by at least 19 mmHg. In a particular embodiment, the method relates to reducing the patient's mRAP by at least 20 mmHg.

[0152] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's mRAP by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to reducing the patient's mRAP by at least 1%. In some embodiments, the method relates to reducing the patient's mRAP by at least 5%. In some embodiments, the method relates to reducing the patient's mRAP by at least 10%. In some embodiments, the method relates to reducing the patient's mRAP by at least 15%. In some embodiments, the method relates to reducing the patient's mRAP by at least 20%. In some embodiments, the method relates to reducing the patient's mRAP by at least 25%. In some embodiments, the method relates to reducing the patient's mRAP by at least 30%. In some embodiments, the method relates to reducing the patient's mRAP by at least 35%. In some embodiments, the method relates to reducing the patient's mRAP by at least 40%. In some embodiments, the method relates to reducing the patient's mRAP by at least 45%. In some embodiments, the method relates to reducing the patient's mRAP by at least 50%. In some embodiments, the method relates to reducing the patient's mRAP by at least 55%. In some embodiments, the method relates to reducing the patient's mRAP by at least 60%. In some embodiments, the method relates to reducing the patient's mRAP by at least 65%. In some embodiments, the method relates to reducing the patient's mRAP by at least 70%. In some embodiments, the method relates to reducing the patient's mRAP by at least 75%.In some embodiments, the method relates to reducing the patient's mRAP by at least 80%. In some embodiments, the method relates to reducing the patient's mRAP by at least 85%. In some embodiments, the method relates to reducing the patient's mRAP by at least 90%. In some embodiments, the method relates to reducing the patient's mRAP by at least 95%. In some embodiments, the method relates to reducing the patient's mRAP by at least 100%.

[0153] PVR In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of PAH, comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has a pulmonary vascular resistance (PVR) of at least 2.5 Wood units (e.g., at least 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 Wood units). In some embodiments, the method relates to a patient having a PVR of at least 2.5 Wood units. In some embodiments, the method relates to a patient having a PVR of at least 3 Wood units. In some embodiments, the method relates to a patient having a PVR of at least 4 Wood units. In some embodiments, the method relates to a patient having a PVR of at least 5 Wood units. In some embodiments, the method relates to a patient having a PVR of at least 6 Wood units. In some embodiments, the method relates to a patient having a PVR of at least 7 Wood units. In some embodiments, the method relates to a patient having a PVR of at least 8 wood units. In some embodiments, the method relates to a patient having a PVR of at least 9 wood units. In some embodiments, the method relates to a patient having a PVR of at least 10 wood units. In some embodiments, the method relates to a patient having a PVR of at least 12 wood units. In some embodiments, the method relates to a patient having a PVR of at least 14 wood units. In some embodiments, the method relates to a patient having a PVR of at least 16 wood units. In some embodiments, the method relates to a patient having a PVR of at least 18 wood units. In some embodiments, the method relates to a patient having a PVR of at least 20 wood units.

[0154] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's PVR. In some embodiments, the reduction of the patient's PVR is a result of a decrease in the patient's mean pulmonary artery pressure (mPAP). In some embodiments, the method relates to reducing the patient's PVR by at least 0.5 Wood units. In some embodiments, the method relates to reducing the patient's PVR by at least 1 Wood unit. In some embodiments, the method relates to reducing the patient's PVR by at least 2 Wood units. In some embodiments, the method relates to reducing the patient's PVR by at least 4 Wood units. In some embodiments, the method relates to reducing the patient's PVR by at least 6 Wood units. In some embodiments, the method relates to reducing the patient's PVR by at least 8 Wood units. In some embodiments, the method relates to reducing the patient's PVR by at least 10 Wood units.

[0155] In some embodiments, the disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's PVR by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%). In some embodiments, the method relates to reducing the patient's PVR. In some embodiments, the reduction in the patient's PVR is a result of a reduction in the patient's mean pulmonary artery pressure (mPAP). In some embodiments, the method relates to reducing the patient's PVR by at least 1%. In some embodiments, the method relates to reducing the patient's PVR by at least 5%. In some embodiments, the method relates to reducing the patient's PVR by at least 10%. In some embodiments, the method relates to reducing the patient's PVR by at least 15%. In some embodiments, the method relates to reducing the patient's PVR by at least 20%. In some embodiments, the method relates to reducing the patient's PVR by at least 25%. In some embodiments, the method relates to reducing the patient's PVR by at least 30%. In some embodiments, the method relates to reducing the patient's PVR by at least 35%. In some embodiments, the method relates to reducing the patient's PVR by at least 40%. In some embodiments, the method relates to reducing the patient's PVR by at least 45%. In some embodiments, the method relates to reducing the patient's PVR by at least 50%. In some embodiments, the method relates to reducing the patient's PVR by at least 55%. In some embodiments, the method relates to reducing the patient's PVR by at least 60%. In some embodiments, the method relates to reducing the patient's PVR by at least 65%. In some embodiments, the method relates to reducing the patient's PVR by at least 70%.In some embodiments, the method relates to reducing the patient's PVR by at least 75%. In some embodiments, the method relates to reducing the patient's PVR by at least 80%. In some embodiments, the method relates to reducing the patient's PVR by at least 85%. In some embodiments, the method relates to reducing the patient's PVR by at least 90%. In some embodiments, the method relates to reducing the patient's PVR by at least 95%. In some embodiments, the method relates to reducing the patient's PVR by at least 100%.

[0156] In some embodiments, PVR is tested after the patient has received a 4-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received an 8-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 12-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 16-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 20-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 22-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 24-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 26-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 28-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, PVR is tested after the patient has received a 48-week treatment using the ActRII polypeptide disclosed herein.

[0157] BNPBoth BNP and NT-proBNP are markers of atrial and ventricular distension resulting from increased intracardiac pressure. The New York Heart Association (NYHA) has developed a four-stage functional classification system for congestive heart failure (CHF) based on the severity of symptoms. Studies have demonstrated that measured concentrations of circulating BNP and NT-proBNP increase with the severity of CHF based on the NYHA classification. In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has a brain natriuretic peptide (BNP) level of at least 100 pg / mL (e.g., at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the method relates to a patient having a BNP level of at least 100 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 150 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 200 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 300 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 400 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 500 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 600 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 700 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 800 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 900 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 1000 pg / mL.In some embodiments, the method relates to a patient having a BNP level of at least 3,000 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 5,000 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 10,000 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 15,000 pg / mL. In some embodiments, the method relates to a patient having a BNP level of at least 20,000 pg / mL. In some embodiments, the method relates to the treatment of a patient having elevated BNP levels compared to a healthy patient.

[0158] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's BNP level by at least 10 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 50 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 100 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 200 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 300 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 400 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 500 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 600 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 700 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 800 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 900 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 1000 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level by at least 5000 pg / mL. In some embodiments, the method relates to reducing the patient's BNP level to a normal level. In some embodiments, a normal level corresponds to a level of <100 pg / mL.

[0159] In some embodiments, the method relates to reducing a patient's BNP by at least 5% (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to reducing a patient's BNP by at least 5%. In some embodiments, the method relates to reducing a patient's BNP by at least 10%. In some embodiments, the method relates to reducing a patient's BNP by at least 15%. In some embodiments, the method relates to reducing a patient's BNP by at least 20%. In some embodiments, the method relates to reducing a patient's BNP by at least 25%. In some embodiments, the method relates to reducing a patient's BNP by at least 30%. In some embodiments, the method relates to reducing a patient's BNP by at least 35%. In some embodiments, the method relates to reducing a patient's BNP by at least 40%. In some embodiments, the method relates to reducing a patient's BNP by at least 45%. In some embodiments, the method relates to reducing a patient's BNP by at least 50%. In some embodiments, the method relates to reducing a patient's BNP by at least 55%. In some embodiments, the method relates to reducing a patient's BNP by at least 60%. In some embodiments, the method relates to reducing a patient's BNP by at least 65%. In some embodiments, the method relates to reducing a patient's BNP by at least 70%. In some embodiments, the method relates to reducing a patient's BNP by at least 75%. In some embodiments, the method relates to reducing a patient's BNP by at least 80%. In some embodiments, the method relates to reducing a patient's BNP by at least 85%. In some embodiments, the method relates to reducing a patient's BNP by at least 90%. In some embodiments, the method relates to reducing a patient's BNP by at least 95%. In some embodiments, the method relates to reducing a patient's BNP by at least 100%.

[0160] NT-proBNP In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), wherein the patient has an NT-proBNP level of at least 100 pg / mL (e.g., at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 3000, 5000, 10,000, 15,000, 20,000, 25,000, or 30,000 pg / mL). In some embodiments, the method relates to a patient having an NT-proBNP level of at least 100 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 150 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 200 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 300 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 400 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 500 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 600 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 700 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 800 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 900 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 1000 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 3000 pg / mL. In some embodiments, the method relates to patients having an NT-proBNP level of at least 5000 pg / mL.In some embodiments, the method relates to a patient having an NT-proBNP level of at least 10,000 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 15,000 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 20,000 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 25,000 pg / mL. In some embodiments, the method relates to a patient having an NT-proBNP level of at least 30,000 pg / mL. In some embodiments, the method relates to the treatment of a patient having elevated NT-proBNP levels compared to a healthy patient.

[0161] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's NT-proBNP level. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 10 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 50 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 100 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 200 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 300 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 400 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 500 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 600 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 700 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 800 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 900 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 1000 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 5000 pg / mL.In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 10,000 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 15,000 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 20,000 pg / mL. In some embodiments, the method relates to reducing the patient's NT-proBNP level by at least 25,000 pg / mL.

[0162] In some embodiments, the method relates to reducing a patient's NT-proBNP levels to normal levels and maintaining their normal NT-proBNP levels. In some embodiments, the disclosure relates to a method for maintaining one or more hemodynamic parameters in a PAH patient at normal levels (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to maintaining a patient's NT-proBNP levels at normal levels. In some embodiments, the method relates to maintaining a patient's NT-proBNP levels below 100 pg / mL. In some embodiments, the method relates to maintaining a patient's NT-proBNP levels below 200 pg / mL. In some embodiments, the method relates to maintaining a patient's NT-proBNP levels below 300 pg / mL. In some embodiments, the method relates to maintaining a patient's NT-proBNP levels below 400 pg / mL.

[0163] In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 5% (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 5%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 10%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 15%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 20%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 25%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 30%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 35%. In some embodiments, the method relates to reducing a patient's NT-proBNP by at least 40%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 45%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 50%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 55%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 60%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 65%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 70%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 75%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 80%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 85%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 90%.In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 95%. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 100%. In some embodiments, the method relates to reducing the patient's NT-proBNP level to a normal level. In some embodiments, a normal level of NT-proBNP is <100 pg / ml. In some embodiments, the method relates to reducing the patient's NT-proBNP level to less than 300 ng / L.

[0164] smooth muscle hypertrophy In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of PAH, comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) such that the patient has smooth muscle hypertrophy. In some embodiments, the Disclosure relates to a method for adjusting one or more parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1). In some embodiments, the method relates to reducing smooth muscle hypertrophy in a patient. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 1%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 5%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 10%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 15%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 20%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 25%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 30%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 35%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 40%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 45%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 50%.In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 55%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 60%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 65%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 70%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 75%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 80%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 85%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 90%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 95%. In some embodiments, the method relates to reducing the patient's smooth muscle hypertrophy by at least 100%.

[0165] Pulmonary arteriole muscularization In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate of progression and / or severity of PAH, comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, such that the patient has increased pulmonary arteriole muscle mass. In some embodiments, the Disclosure relates to reducing one or more parameters in PAH patients to more normal levels (e.g., healthy individuals of similar age and sex). The present invention relates to a method for adjusting the pulmonary arterioles to a normal state (compared to a healthy person), comprising administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need. In some embodiments, the method relates to reducing the muscularity of the pulmonary arterioles in the patient. In some embodiments, the method reduces the muscularity of the pulmonary arterioles in the patient by at least 1% (for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 7 The method relates to reducing by 5%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 1%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 5%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 10%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 15%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 20%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 25%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 30%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 35%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 40%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 45%.In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 50%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 55%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 60%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 65%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 70%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 75%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 80%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 85%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 90%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 95%. In some embodiments, the method relates to reducing the muscularity of the patient's pulmonary arterioles by at least 100%.

[0166] Percentage of hospitalizations In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), thereby reducing the patient's hospitalization rate by at least 1% (e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 1%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 2%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 3%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 4%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 5%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 10%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 15%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 20%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 25%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 30%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 35%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 40%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 45%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 50%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 55%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 60%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 65%.In some embodiments, the method relates to reducing the patient hospitalization rate by at least 70%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 75%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 80%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 85%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 90%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 95%. In some embodiments, the method relates to reducing the patient hospitalization rate by at least 100%. In some embodiments, the method reduces the risk of hospitalization for one or more complications associated with PAH.

[0167] Quality of Life In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), thereby increasing the patient's quality of life by at least 1% (e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the method relates to increasing the patient's quality of life by at least 1%. In some embodiments, the method relates to increasing the patient's quality of life by at least 2%. In some embodiments, the method relates to increasing the patient's quality of life by at least 3%. In some embodiments, the method relates to increasing the patient's quality of life by at least 4%. In some embodiments, the method relates to increasing the patient's quality of life by at least 5%. In some embodiments, the method relates to increasing the patient's quality of life by at least 10%. In some embodiments, the method relates to increasing the patient's quality of life by at least 15%. In some embodiments, the method relates to increasing the patient's quality of life by at least 20%. In some embodiments, the method relates to increasing the patient's quality of life by at least 25%. In some embodiments, the method relates to increasing the patient's quality of life by at least 30%. In some embodiments, the method relates to increasing the patient's quality of life by at least 35%. In some embodiments, the method relates to increasing the patient's quality of life by at least 40%. In some embodiments, the method relates to increasing the patient's quality of life by at least 45%. In some embodiments, the method relates to increasing the patient's quality of life by at least 50%.In some embodiments, the method relates to increasing the patient's quality of life by at least 55%. In some embodiments, the method relates to increasing the patient's quality of life by at least 60%. In some embodiments, the method relates to increasing the patient's quality of life by at least 65%. In some embodiments, the method relates to increasing the patient's quality of life by at least 70%. In some embodiments, the method relates to increasing the patient's quality of life by at least 75%. In some embodiments, the method relates to increasing the patient's quality of life by at least 80%. In some embodiments, the method relates to increasing the patient's quality of life by at least 85%. In some embodiments, the method relates to increasing the patient's quality of life by at least 90%. In some embodiments, the method relates to increasing the patient's quality of life by at least 95%. In some embodiments, the method relates to increasing the patient's quality of life by at least 100%.

[0168] In some embodiments, the patient's quality of life is measured using the Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR). In some embodiments, the patient's quality of life is measured using PAH-SYMPACT®. In some embodiments, the patient's quality of life is measured using the Medical Outcome Survey Short Form 36 (SF-36). In some embodiments, the patient's quality of life is measured using EuroQol. In some embodiments, the patient's quality of life is measured using EuroQol-5 Dimension (EQ-5D). In some embodiments, the patient's quality of life is measured using EuroQol-5 Dimension 5 Level (EQ-5D-5L). In some embodiments, the patient's quality of life is measured using the Kansas City Cardiomyopathy Questionnaire (KCCQ).

[0169] ejection fraction In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), wherein the patient has an ejection fraction of less than 10% (e.g., less than 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55%). In some embodiments, the method relates to a patient having an ejection fraction of less than 10%. In some embodiments, the method relates to a patient having an ejection fraction of less than 15%. In some embodiments, the method relates to a patient having an ejection fraction of less than 20%. In some embodiments, the method relates to a patient having an ejection fraction of less than 25%. In some embodiments, the method relates to a patient having an ejection fraction of less than 30%. In some embodiments, the method relates to a patient having an ejection fraction of less than 35%. In some embodiments, the method relates to a patient having an ejection fraction of less than 40%. In some embodiments, the method relates to patients with an ejection fraction of less than 45%. In some embodiments, the method relates to patients with an ejection fraction of less than 50%. In some embodiments, the method relates to patients with an ejection fraction of less than 55%. In some embodiments, the ejection fraction is the right ventricular ejection fraction. In some embodiments, the ejection fraction is the left ventricular ejection fraction. In some embodiments, the ejection fraction is measured using echocardiography. In some embodiments, the patient has a maintained left ventricular ejection fraction.

[0170] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., >50% ejection fraction), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 1%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 5%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 10%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 15%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 20%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 25%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 30%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 35%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 40%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 45%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 50%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 55%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 60%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 65%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 70%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 75%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 80%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 85%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 90%. In some embodiments, the method relates to increasing the patient ejection fraction by at least 95%.In some embodiments, the method relates to increasing the patient's ejection fraction by at least 100%.

[0171] Right ventricular function In certain embodiments, the present disclosure relates to a method for improving or maintaining right ventricular function in PAH, comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof. Improvement or maintenance of right ventricular function can be assessed by a number of echocardiographic measurements. One such quantitative approach for assessing right ventricular function is the measurement of tricuspid annular systolic displacement (TAPSE). TAPSE estimates RV systolic function by measuring the level of systolic displacement of the lateral tricuspid valve annulus toward the apex. Other echocardiographic measurements that can be used to assess maintenance and / or improvement in right ventricular function include, but are not limited to, percentage change in right ventricular area (RVFAC), right ventricular end-diastolic area (RVEDA), right ventricular end-systolic area (RVESA), right ventricular ejection fraction (RVEF), right ventricle-pulmonary (RV-PA) coupling, pulmonary systolic pressure (PASP), tricuspid regurgitation velocity (TRV), and right ventricular hypertrophy.

[0172] TAP SE The tricuspid annular systolic displacement (TAPSE) can be obtained using echocardiography and represents a measure of long-term RV function. TAPSE has previously been shown to correlate well with parameters that estimate overall RV systolic function. A TAPSE of <17 mm suggests high RV systolic dysfunction. In some embodiments, improvement or maintenance of right ventricular function in PAH patients is measured as an increase in TAPSE. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of 20 mm to 28 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of at least 20 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of at least 22 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of at least 24 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of at least 26 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of at least 28 mm. In some embodiments, TAPSE is measured using echocardiography.

[0173] In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE between 16 mm and 30 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE between 18 mm and 28 mm. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE of at least 18 mm. In some embodiments, TAPSE is measured using echocardiography.

[0174] PASP In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of pulmonary arterial hypertension (PAH), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has a pulmonary artery systolic pressure (PASP) of at least 30 mmHg (e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mmHg). In some embodiments, the method relates to a patient having a PASP of at least 30 mmHg. In some embodiments, the method relates to a patient having a PASP of at least 35 mmHg. In some embodiments, the method relates to a patient having a PASP of at least 40 mmHg. In some embodiments, the method relates to a patient having a PASP of at least 45 mmHg. In some embodiments, the method relates to a patient having a PASP of at least 50 mmHg. In some embodiments, the method relates to a patient with a PASP of at least 55 mmHg. In some embodiments, the method relates to a patient with a PASP of at least 60 mmHg. In some embodiments, the method relates to a patient with a PASP of at least 65 mmHg. In some embodiments, the method relates to a patient with a PASP of at least 70 mmHg. In some embodiments, the method relates to a patient with a PASP of at least 75 mmHg. In some embodiments, the method relates to a patient with a PASP of at least 80 mmHg. In some embodiments, PASP is resting PASP. In some embodiments, PASP is determined using tricuspid regurgitation velocity (TRV) and right arterial (RA) pressure. In some embodiments, PASP is given by the following formula: PASP=TRV 2 ×4 + RA pressure It is determined using

[0175] TRV has been shown to correlate with resting and exercise-induced PASP. The pressure gradient between the right ventricle and right atrium is given by a modified Bernoulli equation (Δp=4V). 2 It can be calculated using ).

[0176] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving pulmonary artery systolic pressure (PASP) in the patient. In some embodiments, the method relates to reducing PASP. In some embodiments, the method relates to reducing the patient's PASP by at least 1 mmHg (e.g., at least 1, 2, 3, 5, 7, 10, 12, 15, 20, 25, 30, or 35 mmHg). In some embodiments, the method relates to reducing the patient's PASP by at least 2 mmHg. In some embodiments, the method relates to reducing the patient's PASP by at least 3 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 5 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 7 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 10 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 12 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 15 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 20 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 25 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 30 mmHg. In certain embodiments, the method relates to reducing the patient's PASP by at least 35 mmHg.

[0177] In some embodiments, the disclosure relates to a method for adjusting one or more hemodynamic parameters in a PH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's PASP by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%). In some embodiments, the method relates to reducing the patient's PASP by at least 1%. In some embodiments, the method relates to reducing the patient's PASP by at least 5%. In some embodiments, the method relates to reducing the patient's PASP by at least 10%. In some embodiments, the method relates to reducing the patient's PASP by at least 15%. In some embodiments, the method relates to reducing the patient's PASP by at least 20%. In some embodiments, the method relates to reducing the patient's PASP by at least 25%. In some embodiments, the method relates to reducing the patient's PASP by at least 30%. In some embodiments, the method relates to reducing the patient's PASP by at least 35%. In some embodiments, the method relates to reducing the patient's PASP by at least 40%. In some embodiments, the method relates to reducing the patient's PASP by at least 45%. In some embodiments, the method relates to reducing the patient's PASP by at least 50%. In some embodiments, the method relates to reducing the patient's PASP by at least 55%. In some embodiments, the method relates to reducing the patient's PASP by at least 60%. In some embodiments, the method relates to reducing the patient's PASP by at least 65%. In some embodiments, the method relates to reducing the patient's PASP by at least 70%. In some embodiments, the method relates to reducing the patient's PASP by at least 75%.In some embodiments, the method relates to reducing the patient's PASP by at least 80%. In some embodiments, the method relates to reducing the patient's PASP by at least 85%. In some embodiments, the method relates to reducing the patient's PASP by at least 90%. In some embodiments, the method relates to reducing the patient's PASP by at least 95%. In some embodiments, the method relates to reducing the patient's PASP by at least 100%.

[0178] RV-PA coupling Right ventricular dysfunction is a central feature of PAH and a major factor affecting prognosis. The energy transfer between ventricular contractility and atrial afterload is called coupling. The specific energy transfer between the right ventricle (RV) and the pulmonary artery is called right ventricular-pulmonary (RV-PA) coupling. In some embodiments, right ventricular dysfunction is attributable to a decrease in RV-PA coupling. RV-PA coupling can be non-invasively estimated as the ratio of TAPSE / PASP values. In some embodiments, a TAPSE / PASP ratio of ≥0.31 mm / mmHg may be associated with a better prognosis and may reduce the risk of clinical deterioration. In some embodiments, improvement in RV-PA coupling is attributable to improvement in PASP. In some embodiments, the calculation of RV-PA coupling depends on the results of a pair of three parameters (e.g., TRV, RAP, and TAPSE).

[0179] In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of pulmonary arterial hypertension (PAH), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has a TAPSE / PASP ratio of less than 0.31 mm / mmHg (e.g., at least 0.3, 0.25, 0.2, 0.15, or 0.1 mm / mmHg). In some embodiments, the method relates to a patient having a TAPSE / PASP ratio of less than 0.31 mm / mmHg. In some embodiments, the method relates to a patient having a TAPSE / PASP ratio of less than 0.3 mm / mmHg. In some embodiments, the method relates to a patient having a TAPSE / PASP ratio of less than 0.3 mm / mmHg. In some embodiments, the method relates to a patient having a TAPSE / PASP ratio of less than 0.25 mm / mmHg. In some embodiments, the method relates to a patient having a TAPSE / PASP ratio of less than 0.2 mm / mmHg. In some embodiments, the method relates to patients having a TAPSE / PASP ratio of less than 0.15 mm / mmHg. In some embodiments, the method relates to patients having a TAPSE / PASP ratio of less than 0.1 mm / mmHg. In some embodiments, the method relates to patients having a reduced TAPSE / PASP ratio compared to a normal TAPSE / PASP ratio.

[0180] In some embodiments, the disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving or maintaining right ventricular function in the patient. In some embodiments, a PAH patient with improved or maintained right ventricular function has a TAPSE / PASP ratio greater than 0.3 mm / mmHg (e.g., greater than 0.31, 0.32, 0.33, 0.34, or 0.35 mm / mmHg). In some embodiments, a PAH patient with improved or maintained right ventricular function has a TAPSE / PASP ratio greater than 0.31 mm / mmHg. In some embodiments, a PAH patient with improved or maintained right ventricular function has a TAPSE / PASP ratio greater than 0.32 mm / mmHg. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE / PASP ratio greater than 0.33 mm / mmHg. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE / PASP ratio greater than 0.34 mm / mmHg. In some embodiments, PAH patients with improved or maintained right ventricular function have a TAPSE / PASP ratio greater than 0.35 mm / mmHg. In some embodiments, improved right ventricular function is an increase in the TAPSE / PASP ratio. In some embodiments, the method relates to increasing the TAPSE / PASP ratio. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.05 mm / mmHg. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.07 mm / mmHg. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.10 mm / mmHg. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.12 mm / mmHg.In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.15 mm / mmHg. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.18 mm / mmHg. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 0.20 mm / mmHg.

[0181] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 5%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 10%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 15%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 20%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 25%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 30%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 35%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 40%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 45%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 50%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 55%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 60%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 65%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 70%.In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 75%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 80%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 85%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 90%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 95%. In some embodiments, the method relates to increasing the patient's TAPSE / PASP ratio by at least 100%.

[0182] RVFAC, RVEDA, and RVESA The percentage change in right ventricular area (RVFAC) is a non-invasive quantitative measure of right ventricular function. RVFAC can be calculated using the formula [(RVEDA-RVESA) / RVEDA]×100. In some embodiments, RVFAC is measured using echocardiography. In some embodiments, a normal RVFAC is approximately 47.5±8.6% in men and approximately 50.9±8.0% in women. See, for example, Kou S, et al. European Heart Journal - Cardiovascular Imaging. 2014 Jun 1;15(6):680-90. In some embodiments, PAH patients have a reduced RVFAC.

[0183] In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of pulmonary arterial hypertension (PAH), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has less than 20% (e.g., less than 20, 25, 30, 35, or 40%) of RVFAC. In some embodiments, the method relates to a patient with less than 25% RVFAC. In some embodiments, the method relates to a patient with less than 30% RVFAC. In some embodiments, the method relates to a patient with less than 35% RVFAC. In some embodiments, the method relates to a patient with less than 40% RVFAC.

[0184] In some embodiments, the disclosure relates to a method for adjusting one or more hemodynamic parameters in a patient with PAH to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving or maintaining right ventricular function in the patient. In some embodiments, the improvement or maintenance of right ventricular function is attributable to an increase in the rate of change of right ventricular area (RVFAC). In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVFAC between 32 and 56%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVFAC of at least 32%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVFAC of at least 34%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVFAC of at least 35%. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 36% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 38% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 40% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 42% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 44% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 46% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 48% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 50% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 52% RVFAC.In some embodiments, PAH patients with improved or maintained right ventricular function have at least 54% RVFAC. In some embodiments, PAH patients with improved or maintained right ventricular function have at least 56% RVFAC.

[0185] In some embodiments, the present disclosure relates to a method for adjusting one or more echocardiographic parameters in a PAH patient to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's RVEDA by at least 1% (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20%). In some embodiments, the method relates to increasing the patient's RVFAC by at least 2%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 3%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 4%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 5%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 6%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 7%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 8%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 9%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 10%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 12%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 14%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 16%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 18%. In some embodiments, the method relates to increasing the patient's RVFAC by at least 20%.

[0186] In some embodiments, the improvement in right ventricular function is due to an increase in ejection fraction. In some embodiments, the improvement in right ventricular function is due to an increase in ejection fraction and an increase in the patient's RVFAC.

[0187] Right ventricular end-diastolic area (RVEDA) can be measured using echocardiography. Normal RVEDA in men is approximately 18.2 ± 4.3 cm². 2 Approximately 14.8 ± 3.5 cm for women. 2 For example, see Kou S, et al. European Heart Journal - Cardiovascular Imaging. 2014 Jun 1;15(6):680-90.

[0188] In certain embodiments, the Disclosure provides a method for treating, preventing, or reducing the rate of progression and / or severity of pulmonary arterial hypertension (PAH), comprising administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has been in contact with at least 22 cm 2 (For example, at least 22, 24, 26, 28, 30, 32, or 34 cm) 2 The present invention relates to a method having RVEDA. In some embodiments, the method has at least 24 cm 2 This relates to patients having RVEDA. In some embodiments, the method is at least 26 cm 2 This relates to patients having RVEDA. In some embodiments, the method is at least 28 cm 2 This relates to patients having RVEDA. In some embodiments, the method is at least 30 cm 2 This relates to patients having RVEDA. In some embodiments, the method is at least 32 cm 2 This relates to patients having RVEDA. In some embodiments, the method is at least 34 cm 2 This relates to patients having RVEDA. In some embodiments, the method relates to patients having elevated RVEDA compared to normal RVEDA.

[0189] In some embodiments, the disclosure relates to a method for adjusting one or more hemodynamic parameters in PAH patients toward more normal levels (e.g., normal compared to a healthy person of similar age and sex), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to patients in need. In some embodiments, the method relates to improving or maintaining right ventricular function in patients. In some embodiments, PAH patients with improved or maintained right ventricular function are 14-22 cm 2 The RVEDA is present. In some embodiments, improvement of right ventricular function is due to a reduction in RVEDA. In some embodiments, the method relates to reducing RVEDA. In some embodiments, the method relates to reducing the patient's RVEDA by at least 1 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 2 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 3 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 4 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 5 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 6 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 7 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 8 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVEDA to at least 9 cm. 2 The method relates to reducing the patient's RVEDA at least 10 cm. 2 Regarding reduction.

[0190] In some embodiments, the present disclosure relates to a method for adjusting one or more echocardiographic parameters in a PAH patient to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's RVEDA by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, or 40%). In some embodiments, the method relates to reducing the patient's RVEDA by at least 5%. In some embodiments, the method relates to reducing the patient's RVEDA by at least 10%. In some embodiments, the method relates to reducing the patient's RVEDA by at least 15%. In some embodiments, the method relates to reducing the patient's RVEDA by at least 20%. In some embodiments, the method relates to reducing the patient's RVEDA by at least 25%. In some embodiments, the method relates to reducing the patient's RVEDA by at least 30%. In some embodiments, the method relates to reducing a patient's RVEDA by at least 35%. In some embodiments, the method relates to reducing a patient's RVEDA by at least 40%.

[0191] The right ventricular end-systolic area (RVESA) can be measured using echocardiography. A normal RVESA in men is approximately 9.6 ± 2.8 cm². 2 Approximately 7.3 ± 2.3 cm for women. 2 For example, see Kou S, et al. European Heart Journal - Cardiovascular Imaging. 2014 Jun 1;15(6):680-90.

[0192] In certain embodiments, the Disclosure provides a method for treating, preventing, or reducing the rate and / or severity of pulmonary arterial hypertension (PAH), comprising administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need thereof, wherein the patient has been in contact with the body for at least 12 cm 2 (For example, at least 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 cm) 2 The present invention relates to a method having RVESA of ) 2 This relates to patients with RVESA. In some embodiments, the method is at least 16 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 18 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 20 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 22 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 24 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 26 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 28 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 30 cm 2 This relates to patients having RVESA. In some embodiments, the method is at least 32 cm 2 This relates to patients having RVESA. In some embodiments, the method relates to patients having increased RVESA compared to normal RVESA.

[0193] In some embodiments, the present disclosure relates to a method for adjusting one or more hemodynamic parameters in PAH patients toward more normal levels (e.g., normal compared to healthy individuals of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving or maintaining right ventricular function in the patient. In some embodiments, PAH patients with improved or maintained right ventricular function are 7-20 cm 2 The patient has a RVESA of at least 1 cm. In some embodiments, improvement in right ventricular function is due to a reduction in RVESA. In some embodiments, the method relates to reducing RVESA. In some embodiments, the method relates to reducing the patient's RVESA by at least 1 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA by at least 2 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA by at least 3 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA by at least 4 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA by at least 5 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA to at least 6 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA by at least 7 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA by at least 8 cm. 2 Regarding reduction. In some embodiments, the method involves reducing the patient's RVESA to at least 9 cm. 2 The method relates to reducing the patient's RVESA by at least 10 cm. 2 Regarding reduction.

[0194] In some embodiments, the present disclosure relates to a method for adjusting one or more echocardiographic parameters in a PAH patient to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's RVESA by at least 1% (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40%). In some embodiments, the method relates to reducing the patient's RVESA by at least 2%. In some embodiments, the method relates to reducing the patient's RVESA by at least 3%. In some embodiments, the method relates to reducing the patient's RVESA by at least 4%. In some embodiments, the method relates to reducing the patient's RVESA by at least 5%. In some embodiments, the method relates to reducing the patient's RVESA by at least 10%. In some embodiments, the method relates to reducing the patient's RVESA by at least 15%. In some embodiments, the method relates to reducing a patient's RVESA by at least 20%. In some embodiments, the method relates to reducing a patient's RVESA by at least 25%. In some embodiments, the method relates to reducing a patient's RVESA by at least 30%. In some embodiments, the method relates to reducing a patient's RVESA by at least 35%. In some embodiments, the method relates to reducing a patient's RVESA by at least 40%.

[0195] RVEF Right ventricular ejection fraction (RVEF) is a comprehensive measure of the systolic performance of the right ventricular cavity (RV). RVEF can be calculated using the RV end-diastolic volume (RVEDV) and RV end-systolic volume (RVESV). Specifically, RVEF can be calculated using the following formula: RVEF(%) = ((RVEDV - RVESV) / ​​RVEDV) × 100. Normal RVEF is approximately 56–65% in men and 60–71% in women. See, for example, Lang RM, J Am Soc Echocardiogr. 2015;28(1):1-39.e14. In some embodiments, RVEF is measured using echocardiography. In some embodiments, the disclosure relates to a method for adjusting one or more hemodynamic parameters in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to improving or maintaining right ventricular function in the patient. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVEF of 45-71%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVEF of 45%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVEF of 50%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVEF of 55%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVEF of 60%. In some embodiments, a PAH patient with improved or maintained right ventricular function has an RVEF of 65%. In some embodiments, PAH patients with improved or maintained right ventricular function have a 70% RVEF.

[0196] In some embodiments, the present disclosure relates to a method for adjusting one or more echocardiographic parameters in a PAH patient to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to increasing the patient's RVEF by at least 2%. In some embodiments, the method relates to increasing the patient's RVEF by at least 3%. In some embodiments, the method relates to increasing the patient's RVEF by at least 4%. In some embodiments, the method relates to increasing the patient's RVEF by at least 5%. In some embodiments, the method relates to increasing the patient's RVEF by at least 6%. In some embodiments, the method relates to increasing the patient's RVEF by at least 7%. In some embodiments, the method relates to increasing the patient's RVEF by at least 8%. In some embodiments, the method relates to increasing the patient's RVEF by at least 9%. In some embodiments, the method relates to increasing the patient's RVEF by at least 10%. In some embodiments, the method relates to increasing the patient's RVEF by at least 11%. In some embodiments, the method relates to increasing the patient's RVEF by at least 12%. In some embodiments, the method relates to increasing the patient's RVEF by at least 13%. In some embodiments, the method relates to increasing the patient's RVEF by at least 14%. In some embodiments, the method relates to increasing the patient's RVEF by at least 15%. In some embodiments, the method relates to increasing the patient's RVEF to a normal level (e.g., 56-65% for men and 60-71% for women).

[0197] right ventricular hypertrophy In certain embodiments, improvement in right ventricular function is measured as a reduction in right ventricular hypertrophy. In some embodiments, right ventricular hypertrophy is measured using the Fulton index (RV / (LV+S)).

[0198] In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of PAH, comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need, wherein the patient has right ventricular hypertrophy. In some embodiments, the Disclosure relates to a method for adjusting one or more parameters in a PAH patient to a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need. In some embodiments, the method relates to reducing right ventricular hypertrophy in a patient. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 1% (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 1%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 5%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 10%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 15%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 20%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 25%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 30%. In some embodiments, the method relates to reducing a patient's right ventricular hypertrophy by at least 35%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 40%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 45%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 50%.In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 55%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 60%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 65%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 70%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 75%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 80%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 85%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 90%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 95%. In some embodiments, the method relates to reducing the patient's right ventricular hypertrophy by at least 100%.

[0199] cardiac output Cardiac output is the volume of blood the heart pumps out per minute. Cardiac output is calculated by multiplying the stroke volume by the heart rate. Generally, a normal resting cardiac output is approximately 4–8 L / min. The cardiac index (CI) is an assessment of cardiac output based on the patient's size. To determine the CI, the cardiac output is divided by the person's body surface area (BSA). The normal range for CI is 2.5–4 L / min / m². 2 Cardiac output can decrease by almost 40% without deviating from the normal limit, approximately 2.5 L / min / m². 2A low cardiac index of less than 100% typically indicates a disturbance in cardiovascular performance. The cardiac index can be calculated using cardiac output (e.g., cardiac index = cardiac output / body surface area). Stroke volume can also be calculated using cardiac output (e.g., stroke volume = CO / heart rate). In certain embodiments, the disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need, thereby increasing the patient's cardiac output by at least 5% (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). In some embodiments, the method relates to increasing the patient's cardiac output by at least 5%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 10%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 15%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 20%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 25%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 30%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 35%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 40%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 45%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 50%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 55%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 60%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 65%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 70%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 75%.In some embodiments, the method relates to increasing the patient's cardiac output by at least 80%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 85%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 90%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 95%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 100%. In some embodiments, the method relates to increasing the patient's cardiac index to at least 4.2 L / min / m. 2 The method relates to increasing the cardiac output to at least 4 L / min. In some embodiments, the cardiac index is measured at rest. In some embodiments, the method relates to increasing the patient's cardiac output to at least 4 L / min. In some embodiments, cardiac output is measured at rest. In some embodiments, cardiac output is measured using a right heart catheter. In some embodiments, cardiac output is measured by thermodilution. In some embodiments, cardiac output is measured using the Fick method.

[0200] Motor skills (6MWD and BDI) In certain embodiments, the present disclosure relates to a method for increasing exercise capacity in patients with PAH, comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to patients in need. Any suitable measure of exercise capacity can be used. For example, exercise capacity in a 6-minute walk test (6MWT), which measures how far a subject can walk in 6 minutes, i.e., the 6-minute walk distance (6MWD), is frequently used to assess the severity of pulmonary hypertension and disease progression. In certain embodiments, exercise capacity can be measured using the Borg Dyspnea Index (BDI). The BDI is a numerical scale for assessing perceived dyspnea (respiratory discomfort). This measures, for example, the degree of shortness of breath after completion of a 6MWT, where a BDI of 0 indicates no shortness of breath and 10 indicates maximum shortness of breath. In some embodiments, the BDI is measured using the BORG CR10 scale.

[0201] In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), wherein the patient has a 6MWD of less than 550 m (e.g., 550, 500, 450, 440, 400, 380, 350, 300, 250, 200, or less than 150 m). In some embodiments, the method relates to a patient having a 6MWD between 150 and 550 m. In some embodiments, the method relates to a patient having a 6MWD between 100 and 500 m. In some embodiments, the method relates to a patient having a 6MWD between 150 and 500 m. In some embodiments, the method relates to a patient having a 6MWD of at least 100 m. In some embodiments, the method relates to a patient having a 6MWD of at least 150 m. In some embodiments, the method relates to a patient having a 6MWD of less than 550 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 500 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 450 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 440 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 400 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 380 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 350 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 300 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 250 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 200 meters. In some embodiments, the method relates to a patient having a 6MWD of less than 150 meters. In some embodiments, the method relates to increasing the patient's 6MWD to >380 meters. In some embodiments, the method relates to increasing the patient's 6MWD to >440 meters.In some embodiments, the method relates to increasing the patient's 6MWD to up to 500 meters. See, for example, Galie N., et al Euro Heart J. (2016) 37, 67-119.

[0202] In some embodiments, the present disclosure relates to a method for adjusting one or more measures of motor capacity in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to increasing the patient's 6MWD by at least 10 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 20 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 25 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 30 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 40 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 50 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 60 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 70 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 80 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 90 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 100 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 125 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 150 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 175 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 200 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 250 meters. In some embodiments, the method relates to increasing the patient's 6MWD by at least 300 meters.In some embodiments, the method relates to increasing the patient's 6MWD by at least 400 meters. In some embodiments, the 6MWD is tested after the patient has received a 4-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, the 6MWD is tested after the patient has received an 8-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, the 6MWD is tested after the patient has received a 12-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, the 6MWD is tested after the patient has received a 16-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, the 6MWD is tested after the patient has received a 20-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, the 6MWD is tested after the patient has received a 22-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, the 6MWD is tested after the patient has received a 24-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, 6MWD is tested after the patient has received a 26-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, 6MWD is tested after the patient has received a 28-week treatment using the ActRII polypeptide disclosed herein. In some embodiments, 6MWD is tested after the patient has received a 48-week treatment using the ActRII polypeptide disclosed herein.

[0203] In some embodiments, the present disclosure relates to a method for adjusting one or more measures of exercise capacity (e.g., BDI) in a PAH patient toward a more normal level (e.g., normal compared to a healthy person of similar age and sex), comprising administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to the patient in need. In some embodiments, the method relates to reducing the patient's BDI. In some embodiments, the method relates to lowering the patient's BDI by at least 0.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 1 exponential point. In some embodiments, the method relates to lowering the patient's BDI by at least 1.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 2 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 2.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 3 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 3.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 4 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 4.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 5.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 6 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 6.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 7 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 7.5 exponential points. In some embodiments, the method relates to lowering the patient's BDI by at least 8 exponential points.In some embodiments, the method relates to reducing the patient's BDI by at least 8.5 index points. In some embodiments, the method relates to reducing the patient's BDI by at least 9 index points. In some embodiments, the method relates to reducing the patient's BDI by at least 9.5 index points. In some embodiments, the method relates to reducing the patient's BDI by at least 10 index points.

[0204] Echocardiography There are a number of clinical presentation factors, echocardiogram features, and other features that can indicate PAH. In patients suspected of having PAH, echocardiograms can be used to measure, among other things, the chamber sizes in the right atrium and right ventricle regions, the magnitude of tricuspid regurgitation, the left ventricular eccentricity index, and right ventricular contractility. Right ventricular contractility can be determined using several variables, such as right ventricular longitudinal axis systolic strain / strain rate and right ventricular fractional area change, Tei index, and tricuspid annular systolic displacement. See, for example, Galie N., et al Euro Heart J. (2016) 37, 67-119.

[0205] In patients with PAH symptoms, echocardiography can be used to evaluate various parameters. For example, in some embodiments, echocardiography can be used to measure the tricuspid annular systolic displacement (TAPSE). In some embodiments, echocardiography can be used to measure pulmonary artery systolic pressure (PASP). In some embodiments, echocardiography can be used to measure tricuspid regurgitation velocity (TRV). In some embodiments, echocardiography can be used to measure the rate of change of right ventricular area (RVFAC). In some embodiments, echocardiography can be used to measure the right ventricular end-systolic area (RVESA). In some embodiments, echocardiography can be used to measure the right ventricular end-diastolic area (RVEDA). In some embodiments, echocardiography can be used to measure the right ventricular ejection fraction (RVEF). In some embodiments, echocardiography can be used to measure the right ventricular stroke volume (RVSV). In some embodiments, echocardiography can be used to measure the left ventricular ejection fraction (LVEF).

[0206] Complications of PAH In certain embodiments, the Disclosure relates to a method for treating, preventing, or reducing the rate and / or severity of progression of one or more complications of PAH, comprising administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need. In some embodiments, the method relates to treating, preventing, or reducing the rate and / or severity of progression of cell proliferation in the pulmonary arteries of a PAH patient. In some embodiments, the method relates to treating, preventing, or reducing the rate and / or severity of progression of smooth muscle and / or endothelial cell proliferation in the pulmonary arteries of a PAH patient. In some embodiments, the method relates to treating, preventing, or reducing the rate and / or severity of progression of angiogenesis in the pulmonary arteries of a PAH patient. In some embodiments, the method relates to increasing the physical activity of a patient with PAH. In some embodiments, the method relates to treating, preventing, or reducing the rate and / or severity of progression of dyspnea in a PAH patient. In some embodiments, the method relates to treating, preventing, or reducing the rate of progression and / or severity of chest pain in PAH patients. In some embodiments, the method relates to treating, preventing, or reducing the rate of progression and / or severity of fatigue in PAH patients. In some embodiments, the method relates to treating, preventing, or reducing the rate of progression and / or severity of pulmonary fibrosis in PAH patients. In some embodiments, the method relates to treating, preventing, or reducing the rate of progression and / or severity of fibrosis in PAH patients. In some embodiments, the method relates to treating, preventing, or reducing the rate of progression and / or severity of pulmonary vascular remodeling in PAH patients. In some embodiments, the method relates to treating, preventing, or reducing the rate of progression and / or severity of cardiac remodeling in PAH patients.In some embodiments, the method relates to treating, preventing, or reducing the rate and / or severity of right ventricular hypertrophy in PAH patients. In some embodiments, the method relates to treating, preventing, or reducing the rate and / or severity of metabolic syndrome in PAH patients.

[0207] Complications or comorbidities In some embodiments, the Disclosure envisions a method for treating one or more complications of PAH (e.g., proliferation of smooth muscle and / or endothelial cells in the pulmonary arteries, angiogenesis in the pulmonary arteries, dyspnea, chest pain, pulmonary vascular remodeling, cardiac remodeling, right ventricular hypertrophy, pulmonary fibrosis, need for lung and / or heart transplantation, and need for atrial septal dehiscence), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who requires it. In some embodiments, the Disclosure envisions a method for preventing one or more complications of PAH, comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who requires it. In some embodiments, the disclosure envisions a method for reducing the rate of progression of one or more complications of PAH, comprising the step of administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need.

[0208] In some embodiments, the Disclosure envisions a method for treating one or more comorbidities of PAH (e.g., systemic hypertension, reduced renal function, diabetes mellitus, obesity, coronary artery disease (CAD), heart failure, and anemia) that comprises the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient in need. In some embodiments, the method results in improvement of one or more comorbidities of PAH (e.g., systemic hypertension, reduced renal function, diabetes mellitus, obesity, coronary artery disease (CAD), heart failure, and anemia). In some embodiments, one or more comorbidities of PAH are indirectly improved (e.g., due to improvement in the patient's PH).

[0209] In some embodiments, the Disclosure intends a method for reducing the rate of progression of PAH, comprising the step of administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who needs it. In some embodiments, the Disclosure intends a method for reducing the severity of PAH, comprising the step of administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who needs it. In some embodiments, the Disclosure intends a method for reducing the need to initiate treatment with known treatments for PAH, comprising the step of administering an effective amount of ActRII polypeptide (for example, an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who needs it. In some embodiments, the Disclosure intends to provide a method for reducing the need to increase the dose of prostacyclin in a patient (e.g., by increasing the dose by at least 10%), comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who needs it. In some embodiments, the Disclosure intends to provide a method for reducing the need for PAH-specific hospitalization, comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who needs it. In some embodiments, PAH-specific hospitalization is the hospitalization of a patient for at least 24 hours. In some embodiments, the Disclosure intends to provide a method for reducing PAH exacerbations, comprising the step of administering an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) to a patient who needs it. In some embodiments, an exacerbation of PAH includes a worsening of the WHO function class and / or a reduction of at least 15% of the patient's 6MWD.

[0210] In some embodiments, a patient receiving one or more ActRII polypeptides disclosed herein (e.g., an amino acid sequence at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1) may require a lower dose or discontinuation of one or more PAH therapies administered concurrently with the ActRII polypeptides. For example, if a patient is receiving one or more ActRII polypeptides in combination with one or more therapies for PAH (e.g., prostacyclin), and the patient shows signs of overdose of the one or more PAH therapies (e.g., prostacyclin), the patient may require a reduced dose of the one or more PAH therapies (e.g., prostacyclin). For example, prostacyclin dilates the pulmonary circulation as well as the systemic circulation, and unnecessary vasodilation can be harmful to the patient. Patients who overdose on prostacyclin typically exhibit an excessively high resting cardiac output. In some embodiments, the dose of one or more PAH therapies (e.g., prostacyclin) may be 4 L / m / m² if the patient is experiencing an overdose of 4 L / m² 2 The resting cardiac index will be reduced based on repeated cardiac output and hemodynamic measurements until it reaches less than 1.0

[0211] In some embodiments, the disclosure envisions a method for reducing the required dose of one or more therapies for PAH in a patient (e.g., reducing the patient's dose by at least 10%), comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1). In some embodiments, the disclosure envisions a method for reducing the required dose of prostacyclin in a patient (e.g., reducing the patient's dose by at least 10%), comprising the step of administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1). In some embodiments, the required dose of one or more therapies for PAH in a patient receiving an effective amount of ActRII polypeptide is reduced by at least 10% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 20%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 30%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 40%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 50%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 60%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 70%.In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 80%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by at least 90%. In some embodiments, the required dose of one or more therapies for PAH in patients receiving an effective dose of ActRII polypeptide is reduced by 100%.

[0212] In some embodiments, one or more therapies for PAH are one or more therapies for PAH disclosed herein. In some embodiments, one or more therapies for PAH are selected from the group consisting of phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptor agonists, and endothelin receptor antagonists. In some embodiments, one or more therapies for PAH are selected from the group consisting of bosentan, sildenafil, beraprost, macitentan, selexipag, epoprostenol, treprostinil, iloprost, ambrisentan, and tadalafil.

[0213] transplant-free survival Lung and / or heart transplantation is a surgical treatment option for patients with PAH and is often recommended for patients who do not respond to less invasive therapies (e.g., vasodilator therapy). Generally, PAH patients undergoing lung and / or heart transplantation have functional class III or class IV pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension.

[0214] In certain embodiments, the Disclosure provides a method for treating, preventing, or reducing the rate and / or severity of PAH, comprising administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), thereby reducing the patient's transplant-free survival by at least 1% (e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%). The present invention relates to a method for increasing by 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 1%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 2%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 3%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 4%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 5%. The method relates to increasing the patient's transplant-free survival by at least 10%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 15%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 20%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 25%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 30%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 35%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 40%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 45%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 50%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 55%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 60%.In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 65%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 70%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 75%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 80%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 85%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 90%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 95%. In some embodiments, the method relates to increasing the patient's transplant-free survival by at least 100%. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than one year compared to a control. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than two years compared to a control. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than three years compared to a control. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than four years compared to a control. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than five years compared to a control. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than 6 years compared to a control. In some embodiments, the method relates to increasing the patient's transplant-free survival by more than 7 years compared to a control.

[0215] death In certain embodiments, the Disclosure relates to a method for reducing the risk of death in patients with PAH, comprising administering to a patient in need an effective amount of ActRII polypeptide (e.g., an amino acid sequence that is at least 90% identical to the amino acid sequence corresponding to residues 30-110 of SEQ ID NO: 1), thereby reducing the patient's risk of death by at least 1% (e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the method relates to reducing the patient's risk of death by at least 1%. In some embodiments, the method relates to reducing the patient's risk of death by at least 2%. In some embodiments, the method relates to reducing the patient's risk of death by at least 3%. In some embodiments, the method relates to reducing the patient's risk of death by at least 4%. In some embodiments, the method relates to reducing the patient's risk of death by at least 5%. In some embodiments, the method relates to reducing the patient's risk of death by at least 10%. In some embodiments, the method relates to reducing the patient's risk of death by at least 15%. In some embodiments, the method relates to reducing the patient's risk of death by at least 20%. In some embodiments, the method relates to reducing the patient's risk of death by at least 25%. In some embodiments, the method relates to reducing the patient's risk of death by at least 30%. In some embodiments, the method relates to reducing the patient's risk of death by at least 35%. In some embodiments, the method relates to reducing the patient's risk of death by at least 40%. In some embodiments, the method relates to reducing the patient's risk of death by at least 45%. In some embodiments, the method relates to reducing the patient's risk of death by at least 50%. In some embodiments, the method relates to reducing the patient's risk of death by at least 55%. In some embodiments, the method relates to reducing the patient's risk of death by at least 60%. In some embodiments, the method relates to reducing the patient's risk of death by at least 65%.In some embodiments, the method relates to reducing the patient's risk of death by at least 70%. In ...

Claims

1. A composition for treating pulmonary arterial hypertension (PAH) in patients in need, comprising a therapeutically effective amount of ActRIIA fusion protein, wherein the fusion protein is (i) ActRIIA polypeptide containing the amino acid sequence of SEQ ID NO: 2, (ii) An Fc domain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 32, and (iii) comprising a linker domain located between the ActRIIA polypeptide and the Fc domain, Here, the fusion protein is administered in a first dose of 0.3 mg / kg and a second dose of 0.7 mg / kg, the second dose being administered three weeks after the first dose, and here, the administration of the fusion protein reduces the patient's risk of death by at least 50%. composition.

2. The composition according to claim 1, wherein the administration of the fusion protein brings about one or more of the following: a. An increase of at least 30 meters in the patient's 6-minute walk distance (6 MWD) from baseline to week 24; b. A decrease of at least 400 pg / mL in N-terminal pro-B natriuretic peptide (NT-proBNP) levels; c. A reduction of at least 10% in the mean right atrial pressure (mRAP) in the patient; d. Improvement in right ventricular function resulting from an increase in the rate of change of right ventricular area, a decrease in right ventricular hypertrophy, and / or an increase in the ejection fraction; e. A reduction of at least one Wood's unit in pulmonary vascular resistance (PVR) from baseline to week 24; f. An increase of at least 25 meters in the 6-minute walk distance (6 MWD) from baseline to week 24; or g. Prevention or reduction of progression of the functional class of pulmonary hypertension as recognized by the World Health Organization (WHO).

3. The composition according to claim 1 or 2, wherein the linker domain is selected from the group consisting of TGGG (SEQ ID NO: 20), TGGGGG (SEQ ID NO: 18), SGGGG (SEQ ID NO: 19), GGGGS (SEQ ID NO: 22), GGG (SEQ ID NO: 16), GGGG (SEQ ID NO: 17), and SGGG (SEQ ID NO: 21).

4. The composition according to any one of claims 1 to 3, wherein the linker domain is TGGG (SEQ ID NO: 20).

5. The composition according to any one of claims 1 to 4, wherein the Fc domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:

32.

6. The composition according to any one of claims 1 to 5, wherein the Fc domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

32.

7. The composition according to any one of claims 1 to 6, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

32.

8. The composition according to any one of claims 1 to 7, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

23.

9. The composition according to any one of claims 1 to 8, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

23.

10. The composition according to any one of claims 1 to 9, wherein the fusion protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

23.

11. The composition according to any one of claims 1 to 10, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

23.

12. The composition according to claim 1, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

41.

13. The composition according to claim 1, wherein the fusion protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

41.

14. The composition according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

41.

15. The composition according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

41.

16. The composition according to any one of claims 1 to 15, wherein the polypeptide is part of a homodimeric protein complex.

17. The composition according to any one of claims 1 to 16, wherein the polypeptide is glycosylated.

18. The composition according to any one of claims 1 to 17, wherein the polypeptide is bound to one or more ligands selected from the group consisting of activin A, activin B, GDF11, BMP10, GDF8, and BMP6.

19. The composition according to any one of claims 1 to 18, wherein the use of the composition improves the right ventricular function of the patient.

20. The composition according to claim 19, wherein the improvement in right ventricular function is due to an increase in the rate of change of right ventricular area, a decrease in right ventricular hypertrophy, and / or an increase in the ejection fraction.

21. The composition according to claim 20, wherein the patient has pulmonary hypertension of functional class I, class II, class III, or class IV according to the World Health Organization's functional classification system for pulmonary hypertension.

22. The composition according to claim 21, wherein the patient has functional class II pulmonary hypertension according to the World Health Organization's functional classification system for pulmonary hypertension.

23. The composition according to claim 21, wherein the patient has pulmonary hypertension of functional class III according to the World Health Organization's functional classification system for pulmonary hypertension.

24. The composition according to claim 21, wherein the patient has pulmonary hypertension of functional class IV according to the World Health Organization's functional classification system for pulmonary hypertension.

25. The composition according to any one of claims 1 to 24, wherein the use of the composition delays the clinical progression of pulmonary arterial hypertension, according to the World Health Organization's functional classification system for pulmonary hypertension.

26. The composition according to any one of claims 1 to 25, wherein the use of the composition reduces the risk of hospitalization for one or more complications associated with pulmonary arterial hypertension.

27. The composition according to any one of claims 1 to 26, wherein the polypeptide is for administration to the patient using subcutaneous injection.

28. The composition according to any one of claims 1 to 27, wherein the first dose of the ActRIIA fusion protein is administered at a dose of 0.3 mg / kg.

29. The composition according to any one of claims 1 to 27, wherein the second dose of the ActRIIA fusion protein is administered at a dose of 0.7 mg / kg.

30. The composition according to any one of claims 1 to 29, further comprising the step of administering an additional activator and / or supportive therapy to the patient.

31. The composition according to claim 30, wherein the additional activator and / or supportive therapy is selected from the group comprising prostacyclins including intravenous epoprostenol, subcutaneous or intravenous treprostinil and inhaled iloprost; phosphodiesterase 5 inhibitors including sildenafil and tadalafil; and endothelin receptor antagonists including oral bosentan and oral ambrisentan.

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