ACTRII-ALK4 ANTAGONISTS AND METHODS FOR TREATING HEART FAILURE

Abstract

In some aspects, the present disclosure relates to ActRII-ALK4 antagonists and methods of using ActRII-ALK4 antagonists to treat, prevent, or reduce the progression rate and / or severity of heart failure (HF), particularly to treat, prevent, or reduce the progression rate and / or severity of one or more HF-related comorbidities. The present disclosure also provides methods of using ActRII-ALK4 antagonists to treat, prevent, or reduce the progression rate and / or severity of heart failure associated with various conditions, including, but not limited to, heart failure associated with metabolic complications (e.g., diabetes, obesity).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 159,051, filed March 10, 2021, and U.S. Provisional Application No. 63 / 227,149, filed July 29, 2021. The specifications of the foregoing applications are incorporated herein by reference in their entireties. [Background technology]

[0002] Excess weight (e.g., obesity) is a rapidly growing problem worldwide and a major risk factor for cardiovascular disease. Similarly, diabetes affects over 180 million people worldwide, and the number of patients is expected to increase to 300 million by 2025 (Mozaffarian, D. et al., Circulation, 2015, 131:e29-322). Among this growing healthcare problem, obesity-related type 2 diabetes accounts for 90-95% of all diagnosed diabetes in adults (Mozaffarian, D. et al., Circulation, 2015, 131:e29-322).

[0003] Epidemiological and clinical data from the past two decades have led to the recognition that, in addition to myocardial infarction and other atherosclerosis-related cardiovascular events, heart failure is the leading cause of cardiovascular morbidity and mortality in patients with diabetes. The prevalence of type 2 diabetes (i.e., diabetes) has increased by 30% worldwide over the past decade, with the number of affected individuals increasing from 333 million in 2005 to 435 million in 2015. As of 2015, 30.3 million Americans (9.4% of the U.S. population) had diabetes. Heart failure (HF) affects at least 26 million people worldwide and is increasing in prevalence. In the United States alone, an estimated 6.5 million adults have HF.

[0004] Diabetes and heart failure often co-occur, and each disease independently increases the risk of the other. In HF cohorts, including both HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF), the prevalence of diabetes ranges from 10% to 47%. The prevalence of diabetes is higher in patients hospitalized with HF, exceeding 40% in some reports. In diabetic patients, the prevalence of HF ranges from 9% to 22%, which is four times higher than in the general population, and the prevalence is even higher in diabetic patients aged 60 years and older.

[0005] Thus, there is a high unmet need for effective therapies for treating heart failure associated with metabolic complications (e.g., diabetes, obesity). Accordingly, it is an object of the present disclosure to provide methods for treating, preventing, or reducing the rate of progression and / or severity of heart failure, and in particular, methods for treating, preventing, or reducing the rate of progression and / or severity of one or more heart failure-related comorbidities. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mozaffarian,D.et al.,Circulation,2015,131:e29-322 Summary of the Invention [Means for solving the problem]

[0007] As demonstrated herein, ActRII-ALK4 antagonists are effective in treating heart failure. In particular, ActRIIB-ALK4 heterodimer proteins demonstrated cardioprotective effects in a mouse model of diabetic cardiomyopathy, a transgenic leptin receptor-deficient mouse model called db / db. In addition to typical diabetic phenotypes such as hyperglycemia, db / db mice exhibit distinct phenotypes of hypertrophic cardiomyopathy, including distinct LV diastolic dysfunction (e.g., heart failure with preserved ejection fraction (HFpEF) phenotype). For example, the data presented herein show that treatment with ActRIIB-ALK4 heterodimers has a positive effect on complications associated with this heart failure model, including but not limited to diastolic function. Without wishing to be bound by a particular mechanism, the effect of the ActRIIB-ALK4 heterodimer on heart failure is expected to be primarily caused by antagonism of ligand signaling mediated by one or more ligands that bind to the ActRIIB-ALK4 heterodimer protein, including, but not limited to, activin A, activin B, GDF8, GDF11, BMP6, and / or BMP10 (referred to herein as "ActRII-ALK4 ligand" or "ActRII-ALK4 ligand"). Regardless of the mechanism, it is clear from the data presented herein that the ActRIIB-ALK4 heterodimer has a significant positive effect on improving various complications associated with heart failure, and further suggests that other ActRII-ALK4 antagonists may also be useful in treating heart failure associated with metabolic complications (e.g., diabetes, obesity).

[0008] As disclosed herein, the term "ActRII-ALK4 antagonist" refers to a variety of agents that can be used to inhibit signaling by one or more ActRII-ALK4 ligands, including, for example, antagonists that inhibit one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and / or BMP10); antagonists that inhibit one or more ActRII-ALK4 ligand-associated receptors (e.g., ActRIIA, ActRIIB, ALK4, and ALK7); and antagonists that inhibit one or more downstream signaling components (e.g., Smad proteins such as Smad2 and 3). ActRII-ALK4 antagonists for use in accordance with the methods and uses of the present disclosure can take various forms, such as ActRII-ALK4 ligand traps (e.g., soluble ActRIIA or ActRIIB polypeptides, including variants and heteromultimers and homomultimers thereof), ActRII-ALK4 antibody antagonists (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and / or ALK4 antagonists), and the like. These include antibodies that inhibit one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and / or ALK7), small molecule antagonists (e.g., small molecules that inhibit one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and / or ALK7), and nucleotide antagonists (e.g., nucleotide sequences that inhibit one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and / or ALK7).

[0009] In certain aspects, the present disclosure provides ActRII-ALK4 antagonists, including soluble ActRIIB, ActRIIA, ALK4, ALK7, or follistatin polypeptides, that generally antagonize the signaling of ActRII-ALK4 ligands in any process related to heart failure associated with metabolic complications (e.g., diabetes, obesity). The ActRII-ALK4 antagonists of the present disclosure can antagonize one or more ligands of ActRII-ALK4, such as activin A, activin B, GDF8, GDF11, BMP6, or BMP10, and thus can be used to treat heart failure associated with metabolic complications (e.g., diabetes, obesity), or one or more comorbidities of heart failure (e.g., anemia, angina pectoris, arterial hypertension, arthritis, atrial fibrillation, cachexia, cancer, cognitive impairment, coronary artery disease (CAD), erectile dysfunction, and the like. , gout, hypercholesterolemia, hyperkalemia, hyperlipidemia, hypertension, iron deficiency, renal dysfunction, metabolic syndrome, physical deconditioning, potassium disorders, pulmonary disease (e.g., asthma, COPD), sarcopenia, sleep apnea, sleep disorders, and valvular heart disease (e.g., aortic stenosis, aortic regurgitation, mitral regurgitation, tricuspid regurgitation).

[0010] In certain embodiments, the ActRII-ALK4 antagonist used in accordance with the methods and uses disclosed herein (e.g., treating, preventing, or reducing the progression rate and / or severity of heart failure associated with metabolic complications (e.g., diabetes, obesity), or one or more complications of heart failure) is an ActRII-ALK4 ligand trap polypeptide antagonist, including variants thereof and heterodimers and heteromultimers thereof, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and / or an ActRII-ALK4 small molecule antagonist. ActRII-ALK4 ligand trap polypeptides include TGF-β superfamily-related proteins (including variants thereof) capable of binding to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Thus, an ActRII-ALK4 ligand trap generally comprises a polypeptide capable of antagonizing one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). As used herein, the term "ActRII" refers to the type II activin receptor family. This family includes activin receptor type IIA (ActRIIA) and activin receptor type IIB (ActRIIB). In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 ligand trap. In some embodiments, an ActRII-ALK4 ligand trap comprises an ActRIIB polypeptide (including variants thereof), and also has homomultimers (e.g., ActRIIB homodimers) and heteromultimers (e.g., ActRIIB-ALK4 or ActRIIB-ALK7 heterodimers). In some embodiments, the ActRII-ALK4 ligand trap comprises an ActRIIA polypeptide (including variants thereof), as well as homomultimers (e.g., ActRIIA homodimers) and heteromultimers (e.g., ActRIIA-ALK4 or ActRIIA-ALK7 heterodimers).In other embodiments, the ActRII-ALK4 ligand trap comprises a soluble ligand trap protein, including, but not limited to, a follistatin polypeptide and variants thereof. In some embodiments, the ActRII-ALK4 antagonist comprises an ActRII-ALK4 antibody antagonist. In some embodiments, the ActRII-ALK4 antagonist comprises an ActRII-ALK4 small molecule antagonist. In some embodiments, the ActRII-ALK4 antagonist comprises an ActRII-ALK4 polynucleotide antagonist.

[0011] In part, the present disclosure provides a method for treating heart failure associated with diabetic cardiomyopathy, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. The present disclosure also provides a method for treating, preventing, or reducing the rate of progression and / or severity of one or more comorbidities of heart failure associated with diabetic cardiomyopathy, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist.

[0012] In part, the present disclosure provides a method for treating heart failure associated with metabolic complications (e.g., diabetes, obesity), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. The present disclosure also provides a method for treating, preventing, or reducing the rate of progression and / or severity of one or more comorbidities of heart failure associated with metabolic complications (e.g., diabetes, obesity), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist.

[0013] In some embodiments of the present disclosure, the patient is obese. In some embodiments, the patient has diabetes. In some embodiments, the patient has type 1 diabetes. In some embodiments, the patient has type 2 diabetes. In some embodiments, the patient has diabetes caused by heart failure. In some embodiments, the patient has heart failure caused by diabetes. In some embodiments, the patient has diabetic cardiomyopathy. In some embodiments, the patient has hyperglycemia. In some embodiments, the patient has insulin resistance. In some embodiments, the patient has hyperinsulinemia. In some embodiments, the patient has one or more of hyperglycemia, insulin resistance, and hyperinsulinemia. In some embodiments, the patient has cardiac insulin resistance.

[0014] In some embodiments of the present disclosure, the patient is diagnosed with mitochondrial dysfunction, oxidative stress, advanced glycation end products (AGEs), mitochondrial Ca 2+ These patients have metabolic disorders that increase one or more of the following: impaired processing, inflammation, activation of the renin-angiotensin-aldosterone system (RAAS), autonomic neuropathy, endoplasmic reticulum stress, myocardial cell death, and microvascular dysfunction.

[0015] In some embodiments, the disclosed methods improve one or more of hyperglycemia, insulin resistance, and hyperinsulinemia in a patient, hi some embodiments, the methods improve cardiac insulin resistance in a patient.

[0016] In some embodiments, the methods of the present disclosure include assessing a patient's mitochondrial dysfunction, oxidative stress, advanced glycation end products (AGEs), mitochondrial Ca 2+ The present invention ameliorates one or more of impaired processing, inflammation, activation of the renin-angiotensin-aldosterone system (RAAS), autonomic neuropathy, endoplasmic reticulum stress, myocardial cell death, and microvascular dysfunction.

[0017] In some embodiments of the present disclosure, the patient is diagnosed with myocardial triglyceride deposition, increased extracellular volume, hyperinsulinemia due to insulin resistance, impaired microcirculation, deposition of AGEs, impaired insulin metabolic signaling that reduces glucose transporter type 4 (GLUT4) recruitment to the plasma membrane and glucose uptake, and sarcoplasmic reticulum Ca 2+ Decreased pump activity, Ca in cardiac cells 2+ Increased myocardial intracellular Ca levels, abnormal insulin metabolic signaling, decreased insulin-stimulated coronary endothelial nitric oxide (NO) synthase (eNOS) activity and NO production, and 2+ / Ca 2+ Increased sensitization, myoplasmic Ca 2+ and metabolic complications selected from the group consisting of decreased NO uptake, decreased NO bioavailability, hyperglycemia, insulin resistance, and oxidative stress.

[0018] In some embodiments, the disclosed methods provide improved myocardial triglyceride deposition, reduced extracellular volume, reduced hyperinsulinemia due to insulin resistance, reduced microcirculatory impairment, reduced AGE deposition, improved GLUT4 recruitment to the plasma membrane and impaired insulin metabolic signaling that reduces glucose uptake, and improved sarcoplasmic reticulum Ca 2+ Increased pump activity, Ca in cardiac cells 2+ the improvement of one or more metabolic complications contributing to cardiovascular structural remodeling selected from the group consisting of: reduction of cardiovascular function, improvement of abnormal insulin metabolic signaling, increase in insulin-stimulated coronary endothelial NO synthase (eNOS) activity and NO production, reduction of cardiomyocyte Ca2+ / Ca2+ sensitization, increase in myocytosolic Ca2+ uptake, increase in NO bioavailability, improvement of hyperglycemia, improvement of insulin resistance, and improvement of oxidative stress.

[0019] In some embodiments of the present disclosure, the heart failure is heart failure with preserved ejection fraction (HFpEF). In some embodiments, the patient has a left ventricular ejection fraction (LVEF) of 50% or greater. In some embodiments, the patient has normal systolic function.

[0020] In some embodiments of the present disclosure, the patient has shortness of breath, hi some embodiments, the methods of the present disclosure reduce shortness of breath.

[0021] In some embodiments of the present disclosure, the patient has cardiovascular structural remodeling selected from the group consisting of increased vascular intimal thickness, increased vascular stiffness, increased left ventricular (LV) hypertrophy, and increased left atrial enlargement. In some embodiments, the methods of the present disclosure improve cardiovascular structural remodeling in the patient selected from the group consisting of increased vascular intimal thickness, increased vascular stiffness, increased LV hypertrophy, and increased left atrial enlargement.

[0022] In some embodiments of the present disclosure, the patient has LV hypertrophy. In some embodiments, the methods of the present disclosure reduce LV hypertrophy in the patient. In some embodiments, the methods reduce left ventricular hypertrophy in the patient, wherein the left ventricular hypertrophy in the patient is reduced by at least 1% (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0023] In some embodiments, the disclosed methods reduce cardiac filling pressure in a patient, hi some embodiments, the methods improve early diastolic cardiac filling in a patient.

[0024] In some embodiments of the present disclosure, the patient has left atrial enlargement. In some embodiments, the methods of the present disclosure reduce atrial enlargement in the patient. In some embodiments, the methods reduce left atrial enlargement in the patient, wherein the left atrial enlargement in the patient is reduced by at least 1% (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%).

[0025] In some embodiments, the methods of the present disclosure reduce vascular intimal thickness in a patient.

[0026] In some embodiments, the methods of the present disclosure reduce vascular stiffness in a patient.

[0027] In some embodiments of the present disclosure, the patient has an alteration in cardiac ventricular structure selected from the group consisting of LV hypertrophy, increased cardiomyocyte size, cardiomyocyte loss, and decreased LV end-diastolic volume.

[0028] In some embodiments, the disclosed methods improve a change in ventricular structure of the patient's heart selected from the group consisting of LV hypertrophy, increased cardiomyocyte size, cardiomyocyte loss, and decreased LV end-diastolic volume. In some embodiments, the methods reduce cardiomyocyte size in the patient. In some embodiments, the methods prevent cardiomyocyte loss from worsening in the patient. In some embodiments, the methods increase LV end-diastolic volume in the patient.

[0029] In some embodiments of the present disclosure, the patient has an altered atrial structure of the heart selected from the group consisting of left atrial hypertrophy, arrhythmia, atrial dilation, aortic root dilation, and atrial fibrillation, hi some embodiments, the methods of the present disclosure improve the altered atrial structure of the heart in the patient selected from the group consisting of left atrial hypertrophy, arrhythmia, atrial dilation, aortic root dilation, and atrial fibrillation.

[0030] In some embodiments of the present disclosure, the patient has a cardiac functional alteration selected from the group consisting of an altered diastolic cardiac function, an altered systolic cardiac function, and an altered electrical cardiac function. In some embodiments, the methods of the present disclosure improve the patient's cardiac functional alteration selected from the group consisting of an altered diastolic cardiac function, an altered systolic cardiac function, and an altered electrical cardiac function.

[0031] In some embodiments, the patient has altered diastolic function. In some embodiments, the patient has diastolic dysfunction. In some embodiments, the methods of the present disclosure improve diastolic dysfunction in the patient. In some embodiments, the patient has decreased ventricular relaxation and increased filling pressure. In some embodiments, the methods increase ventricular relaxation and decrease filling pressure in the patient. In some embodiments, the patient's diastolic dysfunction is measured by the ratio of early diastolic transmitral flow to early diastolic mitral annular tissue velocity (E / e'). In some embodiments, the patient's E / e' ratio is increased compared to a healthy person of similar age and sex. In some embodiments, the patient's E / e' ratio is less than 8. In some embodiments, the patient's E / e' ratio is between 8 and 15. In some embodiments, the patient's E / e' ratio is greater than 15. In some embodiments, the disclosed methods decrease the patient's E / e' ratio, where the patient's E / e' ratio is decreased 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 patient's E / e' ratio is decreased by at least 1 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 40, 45, or 50). In some embodiments, the methods decrease the patient's E / e' ratio to less than 8.

[0032] In some embodiments, the patient has a normal diastolic dysfunction grade. In some embodiments, the patient's normal diastolic dysfunction grade includes an early diastolic transmitral flow to early diastolic left atrial flow ratio (E / A) of 1-2, an E / e' of <8, a normal left atrial volume index (LAVI), and a deceleration time (DT) of <160 ms compared to a healthy person of similar age and sex. In some embodiments, the patient has a diastolic dysfunction grade of 1. In some embodiments, the patient's Grade 1 diastolic dysfunction includes an E / A ratio of less than 1 due to impaired relaxation, an E / e' of less than 8, a normal or increased LAVI, and an increased deceleration time compared to a healthy person of similar age and sex. In some embodiments, the patient has a diastolic dysfunction grade of 2. In some embodiments, the patient's Grade 2 diastolic dysfunction includes an E / A of 1-2, an E / e' of 8-15, an increased LAVI, and a decreased deceleration time compared to a healthy person of similar age and sex. In some embodiments, the patient has a diastolic dysfunction grade of 3. In some embodiments, the patient's Grade 3 diastolic dysfunction includes E / A>2, E / e' greater than 15, increased LAVI, and very short E deceleration times (<140 ms) due to severely reduced LV compliance and high LV filling pressures compared to healthy individuals of similar age and sex.

[0033] In some embodiments, the disclosed methods improve the patient's diastolic dysfunction grade. In some embodiments, the methods improve the patient's diastolic dysfunction grade from Grade 3 to Grade 2. In some embodiments, the methods improve the patient's diastolic dysfunction grade from Grade 3 to Grade 1. In some embodiments, the methods improve the patient's diastolic dysfunction grade from Grade 3 to normal. In some embodiments, the methods improve the patient's diastolic dysfunction grade from Grade 2 to Grade 1. In some embodiments, the methods improve the patient's diastolic dysfunction grade from Grade 2 to normal. In some embodiments, the methods improve the patient's diastolic dysfunction grade from Grade 1 to normal.

[0034] In some embodiments, the methods of the present disclosure increase LV diastolic function in a patient (e.g., by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%).

[0035] In some embodiments of the present disclosure, the patient has an ejection fraction of at least 50% (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%).

[0036] In some embodiments of the present disclosure, the patient is evaluated for changes in electrical function using electrocardiography. In some embodiments, the patient's changes in electrocardiogram measurements are selected from the group consisting of an increase in P wave duration, PR interval, and QT interval, and a leftward shift in T wave voltage and QRS axis. In some embodiments, the methods of the present disclosure improve the patient's electrocardiogram measurements selected from the group consisting of a decrease in P wave duration, a decrease in PR interval, a decrease in QT interval, an increase in T wave voltage, and a shift in the QRS axis to a normal position.

[0037] In some embodiments of the present disclosure, patients are evaluated for diastolic dysfunction using a diastolic stress test. In some embodiments, the diastolic stress test is performed on a bicycle fixed to a catheterization table. In some embodiments, the diastolic stress test is performed using echocardiography. In some embodiments, patients are evaluated for diastolic dysfunction using a diastolic stress test ...septal e' velocity with exercise < 7 cm / s or lateral e' velocity < 10 cm / s at rest, mean E / e' > 14 or septal E / e' ratio > 15, peak tricuspid regurgitation (TR) velocity with exercise > 2.8 m / s, and a diastolic E / e' ratio > 34 mL / m 2In some embodiments, the disclosed methods involve increasing a patient's septal e' velocity to greater than 7 cm / s or lateral e' velocity to greater than 10 cm / s at rest, decreasing mean E / e' to less than 14 or septal E / e' ratio to less than 15 with exercise, decreasing peak tricuspid regurgitation (TR) velocity to less than 2.8 m / s with exercise, and increasing a left atrial volume index (LAVI) to 34 mL / m 2 Reduce to less than.

[0038] In some embodiments, the disclosed methods reduce the patient's H2FPEF score (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, or 9 points). In some embodiments, the patient is evaluated for heart failure using right heart catheterization. In some embodiments, the patient has a pulmonary capillary wedge pressure (PCWP) of 15 mmHg or greater at rest and / or a PCWP of 25 mmHg or greater during exercise. In some embodiments, the disclosed methods reduce the patient's PCWP at rest to at least below 15 mmHg and / or reduce the PCWP during exercise to at least below 25 mmHg.

[0039] In some embodiments of the present disclosure, the patient has a European Heart Failure Association (EHFA) score of 5 or greater. In some embodiments, an EHFA score of 5 or greater indicates HFpEF. In some embodiments, the patient has an EHFA score of 2-4. In some embodiments, an EHFA score of 2-4 indicates the patient has HFpEF. In some embodiments, the patient has an EHFA score of 1 point or less. In some embodiments, an EHFA score of 1 or less indicates the patient does not have HFpEF.

[0040] In some embodiments of the present disclosure, the patient has one or more major functional EHFA criteria for HFpEF. In some embodiments, the major functional criteria are selected from the group consisting of septal e' velocity <7 cm / s with exercise, lateral e' velocity <10 cm / s at rest, mean E / e' > 14 or septal E / e' ratio > 15, and TR velocity > 2.8 m / s with exercise. In some embodiments, the methods of the present disclosure improve one or more major functional criteria selected from the group consisting of increasing septal e' velocity to > 7 cm / s, increasing lateral e' velocity to > 10 cm / s at rest, decreasing E / e' to < 14 with exercise or septal E / e' ratio < 15, and decreasing TR velocity to < 2.8 m / s with exercise.

[0041] In some embodiments of the present disclosure, the patient has one or more major morphological EHFA criteria for HFpEF. In some embodiments, the major morphological criteria are: LAVI > 34 mL / m for men; 2 and LVMI ≥ 149 g / m 2 and ≥122 g / m in women. 2 and RWT>0.42. In some embodiments, the methods of the present disclosure provide for a LAVI of 34 mL / m 2 and LVMI to less than 149 g / m in men. 2 and 122 g / m for women. 2 and reducing RWT to less than 0.42.

[0042] In some embodiments of the present disclosure, the patient has one or more primary biomarker EHFA criteria for HFpEF. In some embodiments, the primary biomarker criteria are sinus rhythm and NT-proBNP >220 pg / mL and / or BNP >80 pg / mL. In some embodiments, the primary biomarker criteria are atrial fibrillation and NT-proBNP >660 pg / mL and / or BNP >240 pg / mL. In some embodiments, the method improves sinus rhythm, including reducing NT-proBNP to <220 pg / mL and / or reducing BNP to <80 pg / mL. In some embodiments, the method of the present disclosure improves atrial fibrillation, including reducing NT-proBNP to <660 pg / mL and / or reducing BNP to <240 pg / mL.

[0043] In some embodiments of the present disclosure, the patient has one or more minor EHFA criteria for HFpEF. In some embodiments, the patient has one or more minor functional EHFA criteria for HFpEF. In some embodiments, the secondary functional criteria are selected from the group consisting of a mean E / e' of 9-14 and a GLS of <16%. In some embodiments, the methods of the present disclosure improve minor functional criteria, including reducing E / e' to 8 or less and increasing GLS to >16%.

[0044] In some embodiments of the present disclosure, the patient has one or more mild morphological EHFA criteria for HFpEF. In some embodiments, the mild morphological criteria are an LAVI of 29-34 mL / m 2 , and LVMI > 115 g / m in men. 2 , LVMI 95g / m for women 2 , RWT>0.42, and LV wall thickness≧12 mm. In some embodiments, the methods of the present disclosure provide for a LAVI of 34 mL / m 2 and LVMI to less than 115 g / m in men. 2 and LVMI of less than 95 g / m in women. 2and improving one or more minor morphological criteria selected from the group consisting of reducing LV wall thickness to less than 12 mm, reducing RWT to less than 0.42, and reducing LV wall thickness to less than 12 mm.

[0045] In some embodiments of the present disclosure, the patient has one or more minor biomarker EHFA criteria for HFpEF. In some embodiments, the minor biomarker criterion is sinus rhythm with 5-NT-proBNP 125-220 pg / mL and / or BNP 35-80 pg / mL. In some embodiments, the minor biomarker criterion is atrial fibrillation with NT-proBNP 365-660 pg / mL and / or BNP 105-240 pg / mL. In some embodiments, the methods of the present disclosure improve sinus rhythm, including reducing 5-NT-proBNP to <220 pg / mL and / or reducing BNP to <80 pg / mL. In some embodiments, the methods of the present disclosure improve atrial fibrillation, including reducing NT-proBNP to <660 pg / mL and / or reducing BNP to <240 pg / mL.

[0046] In some embodiments, the methods of the disclosure reduce a patient's EHFA score (eg, by 1, 2, 3, 4, 5, 6, 7, or 8 points).

[0047] In some embodiments of the present disclosure, the heart failure is heart failure associated with reduced ejection fraction (HFrEF). In some embodiments, the patient's left ventricular ejection fraction (LVEF) is <40%. In some embodiments, the patient's ejection fraction is less than 45% (e.g., 10, 15, 20, 25, 30, 35, 40, or 45%).

[0048] In some embodiments, the disclosed methods increase the patient's ejection fraction by at least 1% (e.g., 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 patient has systolic dysfunction. In some embodiments, the patient has normal systolic function that progresses to systolic dysfunction in advanced stages of the disease. In some embodiments, the disclosed methods improve systolic function. In some embodiments, the disclosed methods increase the patient's cardiac output (e.g., by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%).

[0049] In some embodiments of the present disclosure, the patient has New York Heart Association (NYHA) Class I HF. In some embodiments, the patient has NYHA Class II HF. In some embodiments, the patient has NYHA Class III HF. In some embodiments, the patient has NYHA Class IV HF.

[0050] In some embodiments, the methods of the disclosure reduce a patient's NYHA class. In some embodiments, the methods reduce a patient's NYHA class from class IV to class III. In some embodiments, the methods reduce a patient's NYHA class from class IV to class II. In some embodiments, the methods reduce a patient's NYHA class from class IV to class I. In some embodiments, the methods reduce a patient's NYHA class from class III to class II. In some embodiments, the methods reduce a patient's NYHA class from class III to class I. In some embodiments, the methods reduce a patient's NYHA class from class II to class I.

[0051] In some embodiments of the present disclosure, the patient has American College of Cardiology Foundation / American Heart Association (ACCF / AHA) Stage A heart failure. In some embodiments, the patient has ACCF / AHA Stage B heart failure. In some embodiments, the patient has ACCF / AHA Stage C heart failure. In some embodiments, the patient has ACCF / AHA Stage D heart failure.

[0052] In some embodiments, the disclosed methods reduce the patient's ACCF / AHA stage. In some embodiments, the methods reduce the patient's ACCF / AHA stage from stage D to stage C. In some embodiments, the methods reduce the patient's ACCF / AHA stage from stage D to stage B. In some embodiments, the methods reduce the patient's ACCF / AHA stage from stage D to stage A. In some embodiments, the methods reduce the patient's ACCF / AHA stage from stage C to stage B. In some embodiments, the methods reduce the patient's ACCF / AHA stage from stage C to stage A. In some embodiments, the methods reduce the patient's ACCF / AHA stage from stage B to stage A.

[0053] In some embodiments of the present disclosure, the patient has Killip classification of HF worsening AMI class I heart failure. In some embodiments, the patient has Killip classification of HF worsening AMI class II heart failure. In some embodiments, the patient has Killip classification of HF worsening AMI class III heart failure. In some embodiments, the patient has Killip classification of HF worsening AMI class IV heart failure.

[0054] In some embodiments, the disclosed methods reduce a patient's Killip classification with HF complicating AMI class. In some embodiments, the methods reduce a patient's Killip class from Class IV to Class III. In some embodiments, the methods reduce a patient's Killip class from Class IV to Class II. In some embodiments, the methods reduce a patient's Killip class from Class IV to Class I. In some embodiments, the methods reduce a patient's Killip class from Class III to Class II. In some embodiments, the methods reduce a patient's Killip class from Class III to Class I. In some embodiments, the methods reduce a patient's Killip class from Class II to Class I.

[0055] In some embodiments of the present disclosure, the patient has one or more of the major Framingham criteria for a diagnosis of HF. In some embodiments, the patient has one or more conditions selected from the group consisting of paroxysmal nocturnal dyspnea or orthopnea, jugular vein distention, rales, radiographic cardiomegaly, acute pulmonary edema, S3 gallop, elevated venous pressure of more than 16 cm of water, transit time of 25 seconds or more, hepatojugular reflex, and weight loss of 4.5 kg or more in 5 days in response to treatment.

[0056] In some embodiments of the present disclosure, the patient has one or more mild Framingham criteria for the diagnosis of HF. In some embodiments, the patient has one or more conditions selected from the group consisting of bilateral ankle edema, nocturnal cough, dyspnea on ordinary exercise, hepatomegaly, pleural effusion, a decrease in vital capacity of one-third of maximum recorded, and tachycardia (heart rate greater than 120 beats per minute).

[0057] In some embodiments of the present disclosure, the patient has at least two major Framingham criteria, hi some embodiments, the patient has at least one major Framingham criterion and at least two minor Framingham criteria.

[0058] In some embodiments, the disclosed methods reduce the number of Framingham criteria for heart failure that a patient has. In some embodiments, the methods reduce the number of major Framingham criteria for heart failure that a patient has. In some embodiments, the methods reduce the number of minor Framingham criteria for heart failure that a patient has.

[0059] In some embodiments of the present disclosure, the patient has one or more conditions selected from the group consisting of typical symptoms, less typical symptoms, specific signs, and less specific signs of HF. In some embodiments, the patient has one or more symptoms selected from the group consisting of shortness of breath, orthopnea, paroxysmal nocturnal dyspnea, decreased exercise tolerance, fatigue, tiredness, increased recovery time after exercise, and ankle swelling. In some embodiments, the patient has one or more less typical symptoms selected from the group consisting of nighttime cough, wheezing, bloating, loss of appetite, confusion (especially in the elderly), depression, palpitations, dizziness, syncope, and respiratory distress.

[0060] In some embodiments of the present disclosure, the patient has one or more symptoms of HF. In some embodiments, the patient has one or more symptoms of HF selected from the group consisting of elevated jugular venous pressure, hepatojugular reflux, a third heart sound (gallop rhythm), and a laterally displaced apical impulse. In some embodiments, the patient has one or more non-specific symptoms of HF. In some embodiments, the patient has one or more non-specific symptoms of HF. In some embodiments, the patient has one or more non-specific symptoms of HF selected from the group consisting of weight gain (>2 kg / week), weight loss (in advanced HF), tissue wasting (cachexia), heart murmur, peripheral edema (ankle, sacrum, scrotum), lung creeping, air entry and decreased sensation at the lung bases (pleural effusion), tachycardia, irregular pulse, tachypnea, Cheyne-Stokes respiration, hepatomegaly, ascites, cool extremities, oliguria, and narrow pulse pressure.

[0061] In some embodiments, the disclosed methods reduce the number of signs and / or symptoms of heart failure that a patient has. In some embodiments, the methods reduce the number of signs of heart failure that a patient has. In some embodiments, the methods reduce the number of symptoms of heart failure that a patient has.

[0062] In some embodiments of the present disclosure, the patient has elevated brain natriuretic peptide (BNP) levels compared to a healthy patient. In some embodiments, the patient has a BNP level of at least 35 pg / mL (e.g., 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the methods of the present disclosure reduce the patient's BNP level by at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, the disclosed methods reduce a patient's BNP levels by at least 5 pg / mL (e.g., 5, 10, 50, 100, 200, 500, 1000, or 5000 pg / mL). In some embodiments, the disclosed methods reduce BNP levels to normal levels (i.e., <100 pg / mL).

[0063] In some embodiments of the present disclosure, the patient has elevated N-terminal proBNP (NT-proBNP) levels compared to healthy patients. In some embodiments, the patient has an NT-proBNP level of at least 10 pg / mL (e.g., 10, 25, 50, 100, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, or 20,000 pg / mL). In some embodiments, the methods of the present disclosure reduce the patient's NT-proBNP levels by at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). In some embodiments, the disclosed methods reduce the patient's NT-proBNP levels by at least 10 pg / mL (e.g., 10, 25, 50, 100, 200, 500, 1000, 5000, 10,000, 15,000, 20,000, or 25,000 pg / mL). In some embodiments, the disclosed methods reduce NT-proBNP levels to normal levels (i.e., <100 pg / mL).

[0064] In some embodiments of the present disclosure, the patient has elevated troponin levels compared to a healthy patient, hi some embodiments, the methods of the present disclosure reduce the patient's troponin levels by at least 1% (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%).

[0065] In some embodiments, the disclosed methods reduce a patient's rate of hospitalization by at least 1% (e.g., 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 disclosed methods reduce a patient's rate of worsening heart failure by at least 1% (e.g., 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 disclosed methods reduce the need for a patient to stay in a hospital. In some embodiments, the disclosed methods reduce the total number of hospital visits for a patient. In some embodiments, the disclosed methods increase the time to first hospitalization for a patient. In some embodiments, the disclosed methods extend the patient's lifespan. In some embodiments, the disclosed methods increase the time between hospital visits. In some embodiments, the disclosed methods reduce the number of recurrent hospital visits.

[0066] In some embodiments, the disclosed methods increase the patient's exercise capacity. In some embodiments, the patient has a 6-minute walk distance of between 150 and 400 meters. In some embodiments, the disclosed methods increase the patient's 6-minute walk distance. In some embodiments, the disclosed methods increase the patient's 6-minute walk distance by at least 10 meters (e.g., by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300 meters, or more than 400 meters).

[0067] In some embodiments, the disclosed methods reduce the patient's Borg Dyspnea Index (BDI). In some embodiments, the disclosed methods reduce 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).

[0068] In some embodiments of the present disclosure, the patient is evaluated for heart failure using echocardiography. In some embodiments, the patient is evaluated for heart failure using cardiac magnetic resonance imaging (CMR). In some embodiments, the patient is evaluated for heart failure using CMR with late gadolinium enhancement (LGE). In some embodiments, the patient is evaluated for one or more conditions selected from the group consisting of LV structure and systolic function (e.g., measured by M-mode in a parasternal short axis view at the papillary muscle level), including, but not limited to, LV wall thickness (LVWT), LV mass (LVM), LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), fractional shortening (FS) (calculated using the formula FS=100%×[(EDD−ESD) / EDD]), LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), ejection fraction (calculated using the formula EF=100%×[(EDV−ESV) / EDV]), hypertrophy index (calculated as the ratio of LVM to LVESV), and relative wall thickness (calculated as the ratio of LVWT to LVESD). In some embodiments, the patient's heart failure is assessed using cardiac imaging selected from the group consisting of multi-gated acquisition (MUGA), chest x-ray, single-photon emission computed tomography (SPECT) and radionucleotide ventriculography, positron emission tomography (PET), coronary angiography, and cardiac computed tomography (CT).

[0069] In some embodiments, the methods of the present disclosure further comprise administering to the patient an additional supportive care or active agent, in some embodiments, the additional supportive care or active agent is selected from the group consisting of an angiotensin-converting enzyme (ACE) inhibitor, a beta-blocker, an angiotensin II receptor blocker (ARB), a mineralocorticoid / aldosterone receptor antagonist (MRA), a glucocorticoid, a statin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, a glucagon-like peptide 1 (GLP-1) receptor agonist, a dipeptidyl peptidase-4 (DPP-4) inhibitor, an implantable cardioverter defibrillator (ICD), an angiotensin receptor neprilysin inhibitor (ARNI), and a diuretic.In some embodiments, the additional active agent and / or supportive care is benazepril, captopril, enalapril, lisinopril, perindopril, ramipril, trandolapril, zofenopril, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, timolol, losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan, azilsartan, sarprisartan, telmisartan, progesterone, eplerenone, and spironolactone, beclomethasone, betamethasone, budesonide, cortisone, deflazacort, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, methylpre ... Nisolone, methylprednisone, prednisone, triamcinolone, finerenone, atorvastatin, fluvastatin, lovastatin, pravastatin, pitavastatin, simvastatin, rosuvastatin, canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, exenatide, liraglutide, lixisenatide, dulaglutide, semaglutide, sitagliptin, Saxa Gliptin, linagliptin, alogliptin, valsartan and sacubitril (neprilysin inhibitors), furosemide, bumetanide, torasemide, bendroflumethiazide, hydrochlorothiazide, metolazone, indapamide, spironolactone / eplerenone, amiloride triamterene, hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFAs), I. f channel inhibitors, and perhexiline.

[0070] In some embodiments of the present disclosure, the patient has a comorbidity selected from the group consisting of age, anemia, angina pectoris, arterial hypertension, arthritis, atrial fibrillation, cachexia, cancer, cognitive impairment, coronary artery disease (CAD), erectile dysfunction, gout, hypercholesterolemia, hyperkalemia, hyperlipidemia, hypertension, iron deficiency, renal dysfunction, metabolic syndrome, poor physical condition, potassium disorders, pulmonary disease (e.g., asthma, COPD), sarcopenia, sleep apnea, sleep disorders, and valvular heart disease (e.g., aortic stenosis, aortic regurgitation, mitral regurgitation, tricuspid regurgitation). In some embodiments, the one or more comorbidities to consider in HF are selected from the group consisting of anemia, atrial fibrillation, coronary artery disease (CAD), and sleep apnea.

[0071] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist comprises an ActRIIA polypeptide. In some embodiments, the ActRII-ALK4 antagonist is a heteromultimer.

[0072] In some embodiments of the present disclosure, the ActRIIA polypeptide begins with any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 366 and includes 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, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196 and 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 in any one of 25, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135.

[0073] In some embodiments of the present disclosure, the ActRIIA 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: 367.

[0074] In some embodiments of the present disclosure, the ActRIIA 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: 368.

[0075] In some embodiments of the present disclosure, the ActRIIA polypeptide is a fusion polypeptide comprising an ActRIIA polypeptide domain and one or more heterologous domains. In some embodiments, the fusion polypeptide is an ActRIIA-Fc fusion polypeptide. In some embodiments, the fusion polypeptide further comprises a linker domain disposed between the ActRIIA polypeptide domain and i) the one or more heterologous domains or ii) the Fc domain. In some embodiments, the linker domain is selected from TGGG (SEQ ID NO: 265), TGGGG (SEQ ID NO: 263), SGGGG (SEQ ID NO: 264), GGGGS (SEQ ID NO: 267), GGG (SEQ ID NO: 261), GGGG (SEQ ID NO: 262), and SGGG (SEQ ID NO: 266).

[0076] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 380.

[0077] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 378.

[0078] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist is a homodimeric polypeptide. In some embodiments, the ActRII-ALK4 antagonist is a heteromultimeric polypeptide. In some embodiments, the heteromultimeric polypeptide comprises an ActRIIA polypeptide and an ALK4 polypeptide. In some embodiments, the heteromultimeric polypeptide comprises an ActRIIA polypeptide and an ALK7 polypeptide.

[0079] In some embodiments of the present disclosure, the ALK4 polypeptide comprises 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 selected from the group consisting of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, and 422.

[0080] In some embodiments of the present disclosure, the ALK7 polypeptide comprises 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 selected from the group consisting of SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, and 134.

[0081] In some embodiments of the present disclosure, the ALK4 polypeptide is a fusion polypeptide comprising an ALK4 polypeptide domain and one or more heterologous domains. In some embodiments, the ALK7 polypeptide is a fusion polypeptide comprising an ALK7 polypeptide domain and one or more heterologous domains. In some embodiments, the fusion polypeptide is an ALK4-Fc fusion polypeptide. In some embodiments, the fusion polypeptide is an ALK7-Fc fusion polypeptide. In some embodiments, the ALK4-Fc fusion polypeptide further comprises a linker domain disposed between the ALK4 polypeptide domain and i) one or more heterologous domains or ii) an Fc domain. In some embodiments, the ALK7-Fc fusion polypeptide further comprises a linker domain disposed between the ALK7 polypeptide domain and i) one or more heterologous domains or ii) an Fc domain. In some embodiments, the linker domain is selected from TGGG (SEQ ID NO: 265), TGGGG (SEQ ID NO: 263), SGGGG (SEQ ID NO: 264), GGGGS (SEQ ID NO: 267), GGG (SEQ ID NO: 261), GGGG (SEQ ID NO: 262), and SGGG (SEQ ID NO: 266).

[0082] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. a) an ALK4-Fc fusion polypeptide; b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14; and c) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14. IIA-Fc fusion polypeptides are ALK4-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; c) ALK4-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. d.) ActRIIA-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and ALK4-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; and e.) an ActRIIA-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, and an ALK4-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0083] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. a) an ALK7-Fc fusion polypeptide; b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14; and c) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14. IIA-Fc fusion polypeptides are ALK7-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; c) ALK7-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. d.) ActRIIA-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and ALK7-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; and e.) an ActRIIA-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, and an ALK7-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0084] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0085] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0086] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. and b) an Fc domain selected from an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0087] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0088] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. and b) an Fc domain selected from an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0089] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0090] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0091] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0092] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27. and b) an Fc domain selected from an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26.

[0093] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27. and b) an ActRIIA-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26.

[0094] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0095] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0096] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0097] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0098] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0099] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0100] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0101] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0102] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0103] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0104] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0105] In some embodiments of the present disclosure, the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0106] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0107] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ActRIIA-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0108] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0109] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0110] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist comprises an ActRIIB polypeptide.

[0111] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist is a heteromultimer.

[0112] In some embodiments of the present disclosure, the ActRIIB polypeptide begins with any one of amino acids 20 to 29 of SEQ ID NO:2 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) and continues with any one of amino acids 109 to 134 of SEQ ID NO:2 (e.g., amino acid residues 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 223, 224, 225, 226, 227, 228, or 229). 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134).

[0113] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises 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 amino acids 29 to 109 of SEQ ID NO:2.

[0114] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises 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 amino acids 25 to 131 of SEQ ID NO:2.

[0115] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises 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 amino acids 20 to 134 of SEQ ID NO:2.

[0116] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 53.

[0117] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 388.

[0118] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 389.

[0119] In some embodiments of the present disclosure, the ActRIIB polypeptide is a fusion polypeptide comprising an ActRIIB polypeptide domain and one or more heterologous domains. In some embodiments, the fusion polypeptide is an ActRIIB-Fc fusion polypeptide. In some embodiments, the fusion polypeptide further comprises a linker domain disposed between the ActRIIB polypeptide domain and the one or more heterologous domains or the Fc domain. In some embodiments, the linker domain is selected from TGGG (SEQ ID NO:265), TGGGG (SEQ ID NO:263), SGGGG (SEQ ID NO:264), GGGGS (SEQ ID NO:267), GGG (SEQ ID NO:261), GGGG (SEQ ID NO:262), and SGGG (SEQ ID NO:266). In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO:5. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO:12.

[0120] In some embodiments of the present disclosure, the ActRIIB polypeptide is selected from the group consisting of A24N, S26T, N35E, E37A, E37D, L38N, R40A, R40K, S44T, L46V, L46I, L46F, L46A, E50K, E50P, E50L, E52A, E52D, E52G, E52H, E52K, E52N, E52P, E52R, E52S, E52T, E52Y, Q53R, Q53K, Q53N, Q53H, D54A, K5 5A, K55D, K55E, K55R, R56A, L57E, L57I, L57R, L57T, L57V, Y60D, Y60F, Y60K, Y60P, R64A, R64H, R64K, R64N, N65A, S67N , S67T, G68R, K74A, K74E, K74F, K74I, K74R, K74Y, W78A, W78Y, L79A, L79D, L79E, L79F, L79H, L79K, L79P, L79R, L79S, L 79T, L79W, D80A, D80F, D80G, D80I, D80K, D80M, D80N, D80R, F82A, F82D, F82E, F82I, F82K, F82L, F82S, F82T, F82W, F82 Y, N83A, N83R, T93D, T93E, T93G, T93H, T93K, T93P, T93R, T93S, T93Y, E94K, Q98D, Q98E, Q98K, Q98R, V99E, V99G, V99K, and comprising one or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of E105N, F108I, F108L, F108V, F108Y, E111D, E111H, E111K, 111N, E111Q, E111R, R112H, R112K, R112N, R112S, R112T, A119P, A119V, G120N, E123N, P129N, P129S, P130A, P130R, and A132N.In some embodiments, the ActRIIB polypeptide comprises one or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of L38N, E50L, E52D, E52N, E52Y, L57E, L57I, L57R, L57T, L57V, Y60D, G68R, K74E, W78Y, L79E, L79F, L79H, L79R, L79S, L79T, L79W, F82D, F82E, F82I, F82K, F82L, F82S, F82T, F82Y, N83R, E94K, and V99G.

[0121] In some embodiments of the present disclosure, an ActRIIB polypeptide comprises an L substitution at a position corresponding to E50 of SEQ ID NO:2. In some embodiments, an ActRIIB polypeptide comprises an N substitution at a position corresponding to L38 of SEQ ID NO:2. In some embodiments, an ActRIIB polypeptide comprises a G substitution at a position corresponding to V99 of SEQ ID NO:2. In some embodiments, an ActRIIB polypeptide comprises an R substitution at a position corresponding to N83 of SEQ ID NO:2. In some embodiments, an ActRIIB polypeptide comprises a T substitution at a position corresponding to F82 of SEQ ID NO:2. In some embodiments, an ActRIIB polypeptide comprises an H substitution at a position corresponding to L79 of SEQ ID NO:2.

[0122] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 276. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 278. In some embodiments, the polypeptide comprises an I substitution at the position corresponding to F82 and an R substitution at the position corresponding to N83 of SEQ ID NO: 2.

[0123] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 279. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 332. In some embodiments, the polypeptide comprises a K substitution at the position corresponding to F82 and an R substitution at the position corresponding to N83 of SEQ ID NO: 2.

[0124] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 333. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 335. In some embodiments, the polypeptide comprises a T substitution at the position corresponding to F82 and an R substitution at the position corresponding to N83 of SEQ ID NO:2.

[0125] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 336. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 338. In some embodiments, the polypeptide comprises a T substitution at a position corresponding to F82 of SEQ ID NO:2.

[0126] In some embodiments of the present disclosure, a polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 339. In some embodiments, a polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 341. In some embodiments, a polypeptide comprises an H substitution at a position corresponding to L79 and an I substitution at a position corresponding to F82 of SEQ ID NO:2.

[0127] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 342. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 344. In some embodiments, the polypeptide comprises an H substitution at a position corresponding to L79 of SEQ ID NO:2.

[0128] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 345. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 347. In some embodiments, the polypeptide comprises an H substitution at a position corresponding to L79 and a K substitution at a position corresponding to F82 of SEQ ID NO:2.

[0129] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 348. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 350. In some embodiments, the polypeptide comprises an L substitution at a position corresponding to E50 of SEQ ID NO:2.

[0130] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 351. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 353. In some embodiments, the polypeptide comprises an N substitution at a position corresponding to L38 and an R substitution at a position corresponding to L79 of SEQ ID NO:2.

[0131] In some embodiments of the present disclosure, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 354. In some embodiments, the polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 356. In some embodiments, the polypeptide comprises a G substitution at the position corresponding to V99 of SEQ ID NO:2.

[0132] In some embodiments of the present disclosure, the ActRIIB polypeptide is a homodimeric polypeptide. In some embodiments, the ActRIIB polypeptide is a heterodimeric polypeptide.

[0133] In some embodiments of the present disclosure, the ActRIIB polypeptide is an amino acid sequence beginning with any one of amino acids 20 to 29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO:2 and ending with any one of amino acids 109 to 134 (e.g., amino acid residues 109, 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 134) of SEQ ID NO:2. and amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more amino acid substitutions at positions in SEQ ID NO:2 selected from the group consisting of L38N, E50L, E52N, L57E, L57I, L57R, L57T, L57V, Y60D, G68R, K74E, W78Y, L79F, L79S, L79T, L79W, F82D, F82E, F82L, F82S, F82T, F82Y, N83R, E94K, and V99G.

[0134] In some embodiments of the present disclosure, the ActRIIB polypeptide comprises an amino acid sequence beginning with any one of amino acids 20 to 29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO:2 and ending with any one of amino acids 109 to 134 (e.g., amino acid residues 109, 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 134) of SEQ ID NO:2, as well as any one of the amino acid sequences L38N, E50L, L60L, L70L, L80L, L90L, L10L, L11L, L12L, L13L, L14L, L15L, L16L, L17L, L18L, L19L, L20L, L21L, L22L, L23L, L24L, L25L, L26L, L27L, L28L, L29L, L30L, L31L, L32L, L33L, L40L, L41L, L42L, L43L, L44L, L45L, L46L, L47L, L48L, L49L, L50L, L51L, L52L, L53L, L54L, L55L, L56L, L57L, L58L, , E52D, E52N, E52Y, L57E, L57I, L57R, L57T, L57V, Y60D, G68R, K74E, W78Y, L79E, L79F, L79H, L79R, L79S, L79T, L79W, F82D, F82E, F82I, F82K, F82L, F82S, F82T, F82Y, N83R, E94K and V99G.

[0135] In some embodiments of the present disclosure, an ActRIIB polypeptide comprises 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 amino acids 29-109 of SEQ ID NO: 2. In some embodiments, an ActRIIB polypeptide comprises 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 amino acids 25-131 of SEQ ID NO: 2. In some embodiments, an ActRIIB polypeptide comprises 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 amino acids 20-134 of SEQ ID NO: 2. In some embodiments, an ActRIIB polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 53. In some embodiments, an ActRIIB polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 388. In some embodiments, the ActRIIB polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 389. In some embodiments, the ActRIIB polypeptide comprises one or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of L38N, E50L, E52D, E52N, E52Y, L57E, L57I, L57R, L57T, L57V, Y60D, G68R, K74E, W78Y, L79E, L79F, L79H, L79R, L79S, L79T, L79W, F82D, F82E, F82I, F82K, F82L, F82S, F82T, F82Y, N83R, E94K, and V99G.

[0136] In some embodiments of the present disclosure, the heteromultimeric polypeptide comprises an ActRIIA polypeptide and an ALK4 polypeptide. In some embodiments, the heteromultimeric polypeptide comprises an ActRIIA polypeptide and an ALK7 polypeptide. In some embodiments, the ALK4 polypeptide comprises 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 selected from the group consisting of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, and 422. In some embodiments, the ALK7 polypeptide comprises 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 selected from the group consisting of SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, and 134.

[0137] In some embodiments of the present disclosure, the ActRIIB polypeptide is a fusion polypeptide comprising an ActRIIB polypeptide domain and one or more heterologous domains. In some embodiments, the ALK4 polypeptide is a fusion polypeptide comprising an ALK4 polypeptide domain and one or more heterologous domains. In some embodiments, the ALK7 polypeptide is a fusion polypeptide comprising an ALK7 polypeptide domain and one or more heterologous domains. In some embodiments, the ActRIIB polypeptide is an ActRIIB-Fc fusion polypeptide. In some embodiments, the ALK4 polypeptide is an ALK4-Fc fusion polypeptide. In some embodiments, the ALK7 polypeptide is an ALK7-Fc fusion polypeptide. In some embodiments, the ActRIIB-Fc fusion polypeptide further comprises a linker domain disposed between the ActRIIB polypeptide domain and one or more heterologous domains or the Fc domain. In some embodiments, the ALK4-Fc fusion polypeptide further comprises a linker domain disposed between the ALK4 polypeptide domain and one or more heterologous domains or the Fc domain. In some embodiments, the ALK7-Fc fusion polypeptide further comprises a linker domain disposed between the ALK7 polypeptide domain and one or more heterologous domains or the Fc domain, hi some embodiments, the linker domain is selected from TGGG (SEQ ID NO:265), TGGGG (SEQ ID NO:263), SGGGG (SEQ ID NO:264), GGGGS (SEQ ID NO:267), GGG (SEQ ID NO:261), GGGG (SEQ ID NO:262), and SGGG (SEQ ID NO:266).

[0138] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. a) an ALK4-Fc fusion polypeptide; b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14; and c) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. IIB-Fc fusion polypeptides include: c) ALK4-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; and b) ALK4-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. d.) ActRIIB-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and ALK4-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; and e.) an ActRIIB-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, and an ALK4-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0139] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. a) an ALK7-Fc fusion polypeptide; b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14; and c) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. IIB-Fc fusion polypeptides include: c) ALK7-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15; and b) ALK7-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. d.) ActRIIB-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and ALK7-Fc fusion polypeptides comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; and e.) an ActRIIB-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, and an ALK7-Fc fusion polypeptide comprising an Fc domain at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0140] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0141] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0142] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0143] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0144] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0145] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0146] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0147] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0148] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and an ALK4-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26.

[0149] In some embodiments of the present disclosure, the heteromultimer comprises: a) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27. and b) an ActRIIB-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and an ALK7-Fc fusion polypeptide comprising an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26.

[0150] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0151] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0152] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0153] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0154] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0155] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0156] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0157] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217, and the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at position 146, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

[0158] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0159] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0160] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0161] In some embodiments of the present disclosure, the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0162] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0163] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and the ActRIIB-Fc fusion polypeptide comprises an Fc domain that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0164] In some embodiments of the present disclosure, the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0165] In some embodiments of the present disclosure, the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 132, a tryptophan at amino acid position 144, and an arginine at amino acid position 435, and the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine ​​at amino acid position 127, a serine at amino acid position 144, an alanine at amino acid position 146, and a valine at amino acid position 185.

[0166] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist is a follistatin polypeptide. In some embodiments, the follistatin polypeptide is an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 390, 391, 392, 393, and 394.

[0167] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist inhibits one or more ligands selected from the group consisting of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ALK4, ActRIIA, and ActRIIB.

[0168] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist is an antibody or a combination of antibodies. In some embodiments, the antibody or combination of antibodies binds to one or more ligands selected from the group consisting of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ALK4, ActRIIA, and ActRIIB. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is selected from the group consisting of galetusumab, trevogumab, stamululumab, domagrozumab, landgrozumab, and bimagrumab.

[0169] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist is a small molecule or combination of small molecules that inhibit one or more ligands selected from the group consisting of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ALK4, ActRIIA, and ActRIIB.

[0170] In some embodiments of the present disclosure, the ActRII-ALK4 antagonist is a polynucleotide or a combination of polynucleotides. In some embodiments, the polynucleotide or combination of polynucleotides inhibits one or more ligands selected from the group consisting of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ALK4, ActRIIA, and ActRIIB. [Brief explanation of the drawings]

[0171] [Figure 1] A diagram showing an alignment of the extracellular domains of human ActRIIB (sequence number 1) and human ActRIIA (sequence number 367) with residues predicted herein to directly contact the ligands shown in boxes, based on a combined analysis of multiple ActRIIB and ActRIIA crystal structures. [Figure 2]

[0023] Figure 1 shows the amino acid sequence of human ActRIIB precursor polypeptide (SEQ ID NO: 2; NCBI Reference Sequence NP_001097.2). The signal peptide is underlined, the extracellular domain is in bold (also referred to as SEQ ID NO: 1), and potential N-linked glycosylation sites are boxed. SEQ ID NO: 2 is used as the wild-type reference sequence for human ActRIIB in this disclosure, and the numbering of variants described herein is based on that of SEQ ID NO: 2. [Figure 3] FIG. 1 shows the amino acid sequence of the human ActRIIB extracellular domain polypeptide (SEQ ID NO: 1). [Figure 4] 1 shows the nucleic acid sequence encoding the human ActRIIB precursor polypeptide. SEQ ID NO: 4 consists of nucleotides 434 to 1972 of the NCBI reference sequence NM_001106.4. [Figure 5] FIG. 1 shows the nucleic acid sequence encoding the human ActRIIB(20-134) extracellular domain polypeptide (SEQ ID NO: 3). [Figure 6]

[0023] Figure 1 shows a multiple sequence alignment of various vertebrate ActRIIB precursor polypeptides (SEQ ID NOS: 358-363) lacking their intracellular domains, a human ActRIIA precursor polypeptide (SEQ ID NO: 364) lacking its intracellular domain, and a consensus ActRII precursor polypeptide (SEQ ID NO: 365). Capital letters in the consensus sequence indicate conserved positions. Lowercase letters in the consensus sequence indicate amino acid residues that are predominant but not universal at that position. [Figure 7] Figure 1 shows a multiple sequence alignment of Fc domains from human IgG isotypes using Clustal2.1. The hinge region is shown with a dotted underline. Double underlines indicate examples of positions engineered in IgG1 (SEQ ID NO: 13) Fc to promote asymmetric chain pairing, as well as corresponding positions for other isotypes IgG4 (SEQ ID NO: 17), IgG2 (SEQ ID NO: 14), and IgG3 (SEQ ID NO: 15). [Figure 8A]1 shows a schematic example of a heteromeric polypeptide complex comprising a variant ActRIIB polypeptide (labeled "X") and either an ALK4 polypeptide (labeled "Y") or an ALK7 polypeptide (labeled "Y"). In the illustrated embodiment, the variant ActRIIB polypeptide is part of a fusion polypeptide comprising the first member ("C1") of the interacting pair, and either the ALK4 polypeptide or the ALK7 polypeptide is part of the fusion polypeptide comprising the second member ("C2") of the interacting pair. Suitable interacting pairs include, for example, heavy and / or light chain immunoglobulin interacting pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker may be disposed between the variant ActRIIB polypeptide, ALK4 polypeptide, or ALK7 polypeptide and the corresponding member of the interacting pair. The first and second members of an interacting pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and they can have the same or different amino acid sequences. See Figure 8A. Alternatively, the interacting pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other over self-association. See Figure 8B. [Figure 8B]1 shows a schematic example of a heteromeric polypeptide complex comprising a variant ActRIIB polypeptide (labeled "X") and either an ALK4 polypeptide (labeled "Y") or an ALK7 polypeptide (labeled "Y"). In the illustrated embodiment, the variant ActRIIB polypeptide is part of a fusion polypeptide comprising the first member ("C1") of the interacting pair, and either the ALK4 polypeptide or the ALK7 polypeptide is part of the fusion polypeptide comprising the second member ("C2") of the interacting pair. Suitable interacting pairs include, for example, heavy and / or light chain immunoglobulin interacting pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker may be disposed between the variant ActRIIB polypeptide, ALK4 polypeptide, or ALK7 polypeptide and the corresponding member of the interacting pair. The first and second members of an interacting pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and they can have the same or different amino acid sequences. See Figure 8A. Alternatively, the interacting pair can be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other over self-association. See Figure 8B. [Figure 9] FIG. 1 shows a multiple sequence alignment of various vertebrate ALK4 proteins and human ALK4 (SEQ ID NOs: 414-420). [Figure 10] Figure 1 shows a multiple sequence alignment of various vertebrate ActRIIA proteins and human ActRIIA (sequence numbers 367, 371-377). [Figure 11A]

[0033] Figure 11A shows two schematic examples of heteromeric protein complexes containing type I and type II receptor polypeptides. Figure 11A shows a heterodimeric protein complex containing one type I receptor fusion polypeptide and one type II receptor fusion polypeptide, which can assemble covalently or non-covalently via multimerization domains contained within each polypeptide chain. The two assembled multimerization domains form an interacting pair, which can be guided or unguided. Figure 11B shows a heterotetrameric protein complex containing two heterodimeric complexes as shown in Figure 11A. Higher order complexes can be envisioned. [Figure 11B]

[0033] Figure 11A shows two schematic examples of heteromeric protein complexes containing type I and type II receptor polypeptides. Figure 11A shows a heterodimeric protein complex containing one type I receptor fusion polypeptide and one type II receptor fusion polypeptide, which can assemble covalently or non-covalently via multimerization domains contained within each polypeptide chain. The two assembled multimerization domains form an interacting pair, which can be guided or unguided. Figure 11B shows a heterotetrameric protein complex containing two heterodimeric complexes as shown in Figure 11A. Higher order complexes can be envisioned. [Figure 12]1 shows a schematic example of a heteromeric protein complex comprising a type I receptor polypeptide (denoted as "I") (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 or ALK7 protein from a human or other species such as those described herein) and a type II receptor polypeptide (denoted as "II") (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB or ActRIIA protein from a human or other species such as those described herein). In the illustrated embodiment, the type I receptor polypeptide is part of a fusion polypeptide comprising the first member ("C1") of the interaction pair, and the type II receptor polypeptide is part of a fusion polypeptide comprising the second member ("C2") of the interaction pair. In each fusion polypeptide, a linker can be disposed between the type I or type II receptor polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair can be guided (asymmetric) pairs, meaning that the members of the pair preferentially associate with each other over self-association, or the interaction pair can be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference and can have the same or different amino acid sequences. Traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, but various engineered Fc domains have been designed as guided (asymmetric) interaction pairs [e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106]. [Figure 13A]1 shows a schematic example of a heteromeric protein complex comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ALK4 protein from a human or other species such as those described herein) and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide that includes a first member ("C1") of an interacting pair, and the ActRIIB polypeptide is part of a fusion polypeptide that includes a second member ("C2") of the interacting pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker may be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair may be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See Figure 13A. Alternatively, the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other over self-association. See Figure 13B. Higher-order complexes may be envisioned. See Figures 13C and 13D. [Figure 13B]1 shows a schematic example of a heteromeric protein complex comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ALK4 protein from a human or other species such as those described herein) and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide that includes a first member ("C1") of an interacting pair, and the ActRIIB polypeptide is part of a fusion polypeptide that includes a second member ("C2") of the interacting pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker may be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair may be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See Figure 13A. Alternatively, the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other over self-association. See Figure 13B. Higher-order complexes may be envisioned. See Figures 13C and 13D. [Figure 13C]1 shows a schematic example of a heteromeric protein complex comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ALK4 protein from a human or other species such as those described herein) and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide that includes a first member ("C1") of an interacting pair, and the ActRIIB polypeptide is part of a fusion polypeptide that includes a second member ("C2") of the interacting pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker may be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair may be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See Figure 13A. Alternatively, the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other over self-association. See Figure 13B. Higher-order complexes may be envisioned. See Figures 13C and 13D. [Figure 13D]1 shows a schematic example of a heteromeric protein complex comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ALK4 protein from a human or other species such as those described herein) and an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species such as those described herein). In the illustrated embodiment, the ALK4 polypeptide is part of a fusion polypeptide that includes a first member ("C1") of an interacting pair, and the ActRIIB polypeptide is part of a fusion polypeptide that includes a second member ("C2") of the interacting pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A):95-106). In each fusion polypeptide, a linker may be disposed between the ALK4 or ActRIIB polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair may be unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See Figure 13A. Alternatively, the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair preferentially associate with each other over self-association. See Figure 13B. Higher-order complexes may be envisioned. See Figures 13C and 13D. [Figure 14] Figure 1 shows the purification of ActRIIA-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak (left lane: molecular weight standard; right lane: ActRIIA-hFc) as visualized by sizing column (upper panel) and Coomassie-stained SDS-PAGE (lower panel). [Figure 15A] Figure 1 shows binding of ActRIIA-hFc to activin (top panel) and GDF-11 (bottom panel) as measured by Biacore™ assay. [Figure 15B] Figure 1 shows binding of ActRIIA-hFc to activin (top panel) and GDF-11 (bottom panel) as measured by Biacore™ assay. [Figure 16A] Figure 1 shows ligand binding kinetics values ​​for homodimeric Fc fusion polypeptides containing variant or unmodified ActRIIB domains as determined by surface plasmon resonance at 37°C. Amino acid numbering is based on SEQ ID NO: 2. ND# indicates the value is undetectable over the concentration range tested. Transient* indicates the value is indeterminate due to the transient nature of the interaction. The control sample is ActRIIB-G1Fc (SEQ ID NO: 5). [Figure 16B] Figure 1 shows ligand binding kinetics values ​​for homodimeric Fc fusion polypeptides containing variant or unmodified ActRIIB domains as determined by surface plasmon resonance at 37°C. Amino acid numbering is based on SEQ ID NO: 2. ND# indicates the value is undetectable over the concentration range tested. Transient* indicates the value is indeterminate due to the transient nature of the interaction. The control sample is ActRIIB-G1Fc (SEQ ID NO: 5). [Figure 17] Figure 1 shows ligand binding kinetics values ​​of homodimeric Fc fusion polypeptides containing variant or unmodified ActRIIB domains as determined by surface plasmon resonance at 37°C. Amino acid numbering is based on SEQ ID NO: 2. ND# indicates the value is undetectable over the concentration range tested. Transient binding * indicates the value is indeterminate due to the transient nature of the interaction. The control sample is ActRIIB-G1Fc (SEQ ID NO: 5). [Figure 18]

[0023] Figure 1 shows ligand binding kinetics values ​​for homodimeric Fc fusion polypeptides containing variant or unmodified ActRIIB domains as determined by surface plasmon resonance at 25°C. ND# indicates values ​​not detectable over the concentration range tested. Amino acid numbering is based on SEQ ID NO: 2. [Figure 19] Figure 1 shows comparative ligand binding data for the ALK4-Fc:ActRIIB-Fc heterodimer protein complex compared to the ActRIIB-Fc homodimer and the ALK4-Fc homodimer. For each protein complex, ligands were ranked by koff (a rate constant that correlates well with ligand signaling inhibition) and listed in descending order of binding affinity (the most strongly binding ligands are listed at the top). On the left, yellow, red, green, and blue lines indicate the magnitude of the off-rate constants. Solid black lines indicate ligands with enhanced or unchanged binding to the heterodimer compared to the homodimer, while dashed red lines indicate substantially reduced binding compared to the homodimer. As shown, the ActRIIB-Fc:ALK4-Fc heterodimer exhibits enhanced binding to activin B compared to either homodimer, retains strong binding to activin A, GDF8, and GDF11 as observed with the ActRIIB-Fc homodimer, and exhibits substantially reduced binding to BMP9, BMP10, and GDF3. Similar to the ActRIIB-Fc homodimer, the heterodimer retains intermediate levels of binding to BMP6. [Figure 20]Figure 1 shows comparative ActRIIB-Fc:ALK4-Fc heterodimer / ActRIIB-Fc:ActRIIB-Fc homodimer IC50 data measured by the A-204 reporter gene assay described herein. The ActRIIB-Fc:ALK4-Fc heterodimer inhibits activin A, activin B, GDF8, and GDF11 signaling pathways, similar to the ActRIIB-Fc:ActRIIB-Fc homodimer. However, ActRIIB-Fc:ALK4-Fc heterodimer inhibition of the BMP9 and BMP10 signaling pathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fc homodimer. These data demonstrate that the ActRIIB:ALK4 heterodimer is a more selective antagonist of activin A, activin B, GDF8, and GDF11 than the corresponding ActRIIB:ActRIIB homodimer. [Figure 21]Figure 1 shows comparative ligand binding data for the ActRIIB-Fc:ALK7-Fc heterodimer protein complex compared to the ActRIIB-Fc homodimer and ALK7-Fc homodimer. For each protein complex, ligands were ranked by koff (a rate constant that correlates well with ligand signaling inhibition) and listed in descending order of binding affinity (the most strongly binding ligands are listed at the top). On the left, yellow, red, green, and blue lines indicate the magnitude of the off-rate constants. Solid black lines indicate ligands with enhanced or unchanged binding to the heterodimer compared to the homodimer, while dashed red lines indicate substantially reduced binding compared to the homodimer. As shown, four of the five ligands with strong binding to the ActRIIB-Fc homodimer (activin A, BMP10, GDF8, and GDF11) show reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, with the exception of activin B, which retains tight binding to the heterodimer. Similarly, three of the four ligands with intermediate binding to the ActRIIB-Fc homodimer (GDF3, BMP6, and especially BMP9) show reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, but increased binding to activin C, making it the second strongest ligand interaction with the entire heterodimer. Finally, activin C and BMP5 unexpectedly bind to the ActRIIB-Fc:ALK7 heterodimer with intermediate strength, despite not binding (activin C) or only weakly binding (BMP5) to the ActRIIB-Fc homodimer. The ligands tested do not bind to the ALK7-Fc homodimer. [Figure 22] FIG. 1 shows a multiple sequence alignment of the ALK7 extracellular domain from various vertebrate species (SEQ ID NOs: 425-430). [Figure 23]ActRIIB-Fc:ALK4-Fc rescued cardiac diastolic dysfunction during LV remodeling in young mice. Both "WT-Vehicle" and "db / db-Vehicle" mice were administered an equal volume of PBS vehicle for 8 weeks. "db / db-ActRIIB-Fc:ALK4-Fc" mice were given ActRIIB-Fc:ALK4-Fc (10 mg / kg) for 8 weeks. E / e' was significantly increased in "db / db-Vehicle" mice (n = 9) compared with "WT-Vehicle" mice (n = 10, p < 0.001). However, in "db / db-ActRIIB-Fc:ALK4-Fc" mice, E / e' was significantly decreased by ActRIIB-Fc:ALK4-Fc treatment (n = 9, p < 0.05). Data are presented as mean ± standard error of the mean. Statistical tests (one-way ANOVA with post-hoc analysis using Tukey's test for multiple comparisons) were performed and the significance level was set as p<0.05, specifically *p<0.05, **p<0.01, ***p<0.001. [Figure 24]ActRIIB-Fc:ALK4-Fc rescued abnormal ECG during left heart remodeling in young mice. In Figures A–C, both "WT-Vehicle" and "db / db-Vehicle" mice were administered an equal volume of PBS vehicle for 8 weeks. "db / db-ActRIIB-Fc:ALK4-Fc" mice were given ActRIIB-Fc:ALK4-Fc (10 mg / kg) for 8 weeks. A. The P amplitude of the positive P wave was significantly increased in "db / db-Vehicle" mice (n = 9) compared with "WT-Vehicle" mice (n = 10, p < 0.01). The P amplitude of the positive P wave was significantly decreased by ActRIIB-Fc:ALK4-Fc treatment in "db / db-ActRIIB-Fc:ALK4-Fc" mice (n = 9, p < 0.05). B. The P amplitude of the negative P wave was significantly increased in db / db-vehicle mice (n = 9) compared with WT-vehicle mice (n = 10, p < 0.05). The P amplitude of the negative P wave tended to decrease in ActRIIB-Fc:ALK4-Fc-treated mice (db / db-ActRIIB-Fc:ALK4-Fc) (n = 9, p > 0.05). Data are presented as mean ± standard error of the mean. Statistical testing (one-way ANOVA with post-hoc analysis using Tukey's test for multiple comparisons) was performed, and the significance level was set at p < 0.05. Specifically, *p < 0.05, **p < 0.01, and ***p < 0.001. [Figure 25]ActRIIB-Fc:ALK4-Fc rescued diastolic dysfunction and abnormal ECG during LV remodeling in aged mice. In Figures A-B, both "WT-vehicle" and "db / db-vehicle" mice were administered an equal volume of PBS vehicle for 8 weeks. "db / db-ActRIIB-Fc:ALK4-Fc" mice were given ActRIIB-Fc:ALK4-Fc (10 mg / kg) for 8 weeks. AE / e' was significantly increased in "db / db-vehicle" mice (n = 10) compared with "WT-vehicle" mice (n = 10, p < 0.0001). However, in "db / db-ActRIIB-Fc:ALK4-Fc" mice, E / e' was significantly decreased by ActRIIB-Fc:ALK4-Fc treatment (n = 9, p < 0.05). BR amplitude was significantly increased in "db / db-vehicle" mice (n = 10) compared with "WT-vehicle" mice (n = 10, p < 0.05). R amplitude was significantly decreased by ActRIIB-Fc:ALK4-Fc treatment in "db / db-ActRIIB-Fc:ALK4-Fc" mice (n = 9, p < 0.01). Data are presented as mean ± standard error of the mean. Statistical tests (one-way ANOVA with post-hoc analysis using Tukey's test for multiple comparisons) were performed, and the significance level was set at p < 0.05. Specifically, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). [Figure 26]ActRIIB-Fc:ALK4-Fc rescued diastolic dysfunction and abnormal ECG during LV remodeling in 14- and 19-week-old db / db mice. In Figures A-B, "middle-aged db / db-vehicle," "aged db / db-vehicle," and age-matched "middle-aged WT-vehicle" and "aged WT-vehicle" mice were administered an equal volume of PBS vehicle for 8 weeks. "middle-aged db / db-ActRIIB-Fc:ALK4-Fc" and "aged db / db-ActRIIB-Fc:ALK4-Fc" mice were administered ActRIIB-Fc:ALK4-Fc (10 mg / kg) for 8 weeks. AE / e' was significantly increased in "middle-aged db / db-vehicle" mice (n = 10) compared with "middle-aged WT-vehicle" mice (n = 10, p < 0.001). However, in "middle-aged db / db-ActRIIB-Fc:ALK4-Fc" mice, ActRIIB-Fc:ALK4-Fc treatment significantly decreased E / e' (n = 10, p < 0.05). E / e' was significantly increased in "aged db / db-vehicle" mice (n = 10) compared with "aged WT-vehicle" mice (n = 10, p < 0.001). However, in "aged db / db-ActRIIB-Fc:ALK4-Fc" mice, ActRIIB-Fc:ALK4-Fc treatment decreased E / e' (n = 9, p > 0.05). BR amplitude was increased in "middle-aged db / db-vehicle" mice (n = 10) compared with "middle-aged WT-vehicle" mice (n = 10, p > 0.05). ActRIIB-Fc:ALK4-Fc treatment significantly reduced R amplitude in "middle-aged db / db-ActRIIB-Fc:ALK4-Fc" mice (n = 10, p < 0.01). R amplitude was increased in "aged db / db-vehicle" mice (n = 10) compared with "aged WT-vehicle" mice (n = 10, p > 0.05). ActRIIB-Fc:ALK4-Fc treatment significantly reduced R amplitude in "aged db / db-ActRIIB-Fc:ALK4-Fc" mice (n = 9, p < 0.01). Data are presented as mean ± standard error of the mean. Statistical testing (one-way ANOVA with post-hoc analysis using Tukey's test for multiple comparisons) was performed, and the significance level was set at p < 0.05.In particular, *p<0.05, **p<0.01, ***p<0.001. [Figure 27] ActRIIB(K55E)-Fc rescued diastolic dysfunction and abnormal ECG during LV remodeling in 19-week-old db / db mice. (Figures A-B) Both "aged WT-vehicle" and "aged db / db-vehicle" mice were administered an equal volume of PBS vehicle for 8 weeks. "aged db / db-ActRIIB(K55E)-Fc" mice were given ActRIIB(K55E)-Fc (10 mg / kg) for 8 weeks. AE / e' was significantly increased in "aged db / db-vehicle" mice (n = 10) compared with "aged WT-vehicle" mice (n = 10, p < 0.0001). However, in "aged db / db-ActRIIB(K55E)-Fc" mice, ActRIIB(K55E)-Fc treatment significantly decreased E / e' (n = 10, p < 0.0001). BR amplitude was significantly increased in "aged db / db-vehicle" mice (n = 10) compared with "aged WT-vehicle" mice (n = 10, p < 0.01). R amplitude was significantly decreased by ActRIIB(K55E)-Fc treatment in "aged db / db-ActRIIB(K55E)-Fc" mice (n = 10, p < 0.05). Data are presented as mean ± standard error of the mean. Statistical testing (one-way ANOVA with post-hoc analysis using Tukey's test for multiple comparisons) was performed, and the significance level was set at p < 0.05. In particular, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 28] Pathophysiology of heart failure in diabetes mellitus. Hyperglycemia, insulin resistance, and hyperinsulinemia, often associated with diabetes mellitus, trigger a cascade of adverse effects that contribute to the development of heart failure in diabetes mellitus. AGEs are advanced glycation end products; CAD is coronary artery disease; LVH is left ventricular hypertrophy; and RAAS is the renin-angiotensin-aldosterone system. (Dunlay, SM, et al., Circulation, 2019, 140:e294-e324) [Figure 29]ActRIIBL79S-Fc rescued diastolic dysfunction during LV remodeling in 14-week-old db / db mice. In Figures A and B, both "WT-Vehicle" and "db / db-Vehicle" mice were administered an equal volume of PBS vehicle for 8 weeks. "db / db-ActRIIBL79S-Fc" mice were given ActRIIBL79S-Fc (10 mg / kg) for 8 weeks. Figure 29A. E / e' was significantly increased in "db / db-Vehicle" mice (n = 10) compared with "WT-Vehicle" mice (n = 10, p < 0.001). However, in "db / db-ActRIIBL79S-Fc" mice, ActRIIBL79S-Fc treatment significantly decreased E / e' (n = 8, p < 0.01). Figure 29B. R amplitude was significantly increased in "db / db-vehicle" mice (n = 10) compared with "WT-vehicle" mice (n = 10, p < 0.01). R amplitude was not modified by ActRIIBL79S-Fc treatment in "db / db-ActRIIBL79S-Fc" mice (n = 8, p > 0.05). Data are presented as mean ± standard error of the mean. Statistical tests (one-way ANOVA with post-hoc analysis using Tukey's test for multiple comparisons) were performed, and the significance level was set at p < 0.05. Specifically, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0172] 1. Overview In certain aspects, the present disclosure relates to a method of using a TGF-β superfamily ligand antagonist, particularly an ActRII-ALK4 antagonist, to treat heart failure. For example, the ActRII-ALK4 antagonists described herein can be used to treat, prevent, or reduce the progression rate and / or severity of heart failure associated with metabolic complications (e.g., diabetes, obesity), or one or more complications of heart failure associated with metabolic complications (e.g., diabetes, obesity).

[0173] Heart failure (HF) is a clinical syndrome characterized by symptoms including shortness of breath, ankle swelling, and fatigue, and may be accompanied by signs including elevated jugular venous pressure, pulmonary clefts, and peripheral edema caused by structural and / or functional cardiac abnormalities. HF typically results in reduced cardiac output and / or elevated intracardiac pressure at rest or during stress.

[0174] Before clinical symptoms become apparent, patients may present with asymptomatic structural or functional cardiac abnormalities (e.g., systolic or diastolic left ventricular (LV) dysfunction) that are precursors to HF. Recognition of these precursors is important because they are associated with poor outcomes, and initiating treatment at the precursor stage may reduce mortality in patients with asymptomatic systolic LV dysfunction.

[0175] Demonstration of the underlying cardiac cause is central to the diagnosis of HF. This usually involves myocardial abnormalities that cause systolic and / or diastolic ventricular dysfunction. However, abnormalities of the valves, pericardium, endocardium, cardiac rhythm, and conduction can also cause HF (and more than one abnormality is often present). Identification of the underlying cardiac problem is important for therapeutic reasons, as the exact pathology determines the specific treatment used (e.g., valve repair or replacement for valvular disease, specific pharmacological therapy for HF with reduced EF, heart rate reduction in tachycardial cardiomyopathy, etc.).

[0176] TGF-β superfamily ligand signaling is mediated by heteromeric complexes of type I and type II serine / threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors are all transmembrane polypeptides, consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signal transduction, while type II receptors are required for ligand binding. Type I and type II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of the type I receptor by the type II receptor.

[0177] Two related type II receptors, ActRIIA and ActRIIB, have been identified as type II receptors for activin (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68:97-108). In addition to activin, ActRIIA and ActRIIB can biochemically interact with several other TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). We have found that soluble ActRIIA-Fc fusion polypeptides and ActRIIB-Fc fusion polypeptides have substantially different effects in vivo, with ActRIIA-Fc having a predominant effect on bone and ActRIIB-Fc having a predominant effect on skeletal muscle.

[0178] Ligands in the TGF-β superfamily share the same dimeric structure, with the central 3.5-turn helix of one monomer facing the concave surface formed by the beta chains of the other monomer. Most TGF-β family members are further stabilized by an intermolecular disulfide bond. This disulfide bond crosses the ring formed by two other disulfide bonds, creating what is called a "cysteine ​​knot" motif [Lin et al. (2006) Reproduction 132:179-190; and Hinck et al. (2012) FEBS Letters 586:1860-1870].

[0179] Activins are members of the TGF-β superfamily, originally discovered as regulators of follicle-stimulating hormone secretion, but have since been characterized for a variety of reproductive and non-reproductive roles. They consist of two closely related β subunits (β A β A、 β B β B , and β A β B There are three major activin forms (A, B, and AB) that are homo / heterodimers of β. The human genome also encodes activin C and activin E, which are expressed primarily in the liver, and β. C or β EHeterodimeric forms containing activin are also known. In the TGF-β superfamily, activin is a unique and multifunctional factor that stimulates hormone production in ovarian and placental cells, supports neuronal cell survival, positively or negatively influences cell cycle progression depending on the cell type, and can induce mesodermal differentiation, at least in amphibian embryos [DePaolo et al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997) Curr Biol. 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963]. In some tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, in the regulation of follicle-stimulating hormone (FSH) secretion from the pituitary gland, activin promotes FSH synthesis and secretion, whereas inhibin reduces FSH synthesis and secretion. Other proteins that may regulate the biological activity of activin and / or bind to activin include follistatin (FS) and α2-macroglobulin.

[0180] As described herein, an agent that binds to "activin A" can be isolated β A Whether in relation to subunits, dimeric complexes (e.g., β A β A Homodimer or β A β B heterodimer) or as β A Subunit-specific binding agents for heterodimeric complexes (e.g., β A β B In the case of activin A, the drug that binds to activin A is A Specific for epitopes present within the subunits, but not the non-β subunits of the complex A subunits (e.g., β of the complex) B Similarly, the agents disclosed herein that antagonize (inhibit) "activin A" do not bind to epitopes present within the isolated β A Whether in association with subunits, dimeric complexes (e.g., βA β A Homodimer or β A β B heterodimer) or as β A An agent that inhibits one or more activities mediated by the β subunit. A β B In the case of heterodimers, drugs that inhibit activin A inhibit β A Specifically inhibits the activity of one or more of the subunits, but not the non-β subunits of the complex. A subunits (e.g., β of the complex) B This principle also applies to agents that bind to and / or inhibit "activin B," "activin C," and "activin E." Agents disclosed herein that antagonize "activin AB" are agents that do not inhibit the activity of β A One or more activities mediated by the subunit and β B An agent that inhibits one or more activities mediated by the subunit.

[0181] BMPs and GDFs together form a cysteine ​​knot cytokine family that shares the characteristic fold of the TGF-β superfamily [Rider et al. (2010) Biochem J., 429(1):1-12]. This family includes, for example, BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4, BMP5, BMP6, BMP7, BMP8, BMP8a, BMP8b, BMP9 (also known as GDF2), BMP10, BMP11 (also known as GDF11), BMP12 (also known as GDF7), BMP13 (also known as GDF6), BMP14 (also known as GDF5), BMP15, GDF1, GDF3 (also known as VGR2), GDF8 (also known as myostatin), GDF9, GDF15, and decapentaplegic. In addition to their ability to induce bone formation, BMPs / GDFs exhibit morphogenetic activity in the development of a wide range of tissues. BMP / GDF homodimers and heterodimers interact with combinations of type I and type II receptor dimers to generate multiple possible signaling complexes, leading to the activation of one of two competing sets of SMAD transcription factors. BMPs / GDFs have highly specific and localized functions. These are regulated in several ways, including the developmental restriction of BMP / GDF expression and the secretion of several specific BMP antagonist proteins that bind cytokines with high affinity. Interestingly, many of these antagonists resemble TGF-β superfamily ligands.

[0182] Growth differentiation factor-8 (GDF8), also known as myostatin, is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in developing and adult skeletal muscle. GDF8 null mutations in transgenic mice are characterized by significant skeletal muscle hypertrophy and hyperplasia (McPherron et al., Nature, 1997, 387:83-90). Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974, Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994, 38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997, 94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and, surprisingly, in humans (Schuelke et al., N Engl J Med 2004;350:2682-8). Studies have also shown that muscle atrophy associated with HIV infection in humans is accompanied by increased expression of the GDF8 polypeptide (Gonzalez-Cadavid et al., Proc Natl Acad Sci USA, 1998, 95:14938-43). Furthermore, GDF8 can regulate the production of muscle-specific enzymes (e.g., creatine kinase) and myoblast proliferation (WO 00 / 43781). The GDF8 propeptide can noncovalently bind to the mature GDF8 domain dimer, inactivating its biological activity (Miyazono et al. (1988) J. Biol. Chem., 263:6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263:7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43).Other polypeptides that bind to and inhibit the biological activity of GDF8 or structurally related polypeptides include follistatin and potentially follistatin-related polypeptides (Gamer et al. (1999) Dev. Biol., 208:222-232).

[0183] Growth differentiation factor-11 (GDF11), also known as BMP11, is a secreted protein (McPherron et al., 1999, Nat. Genet. 22:260-264). GDF11 is expressed in the tail bud, limb bud, upper and lower mandibular arch, and dorsal root ganglion during mouse development (Nakashima et al., 1999, Mech. Dev. 80:185-189). GDF11 plays a unique role in the patterning of both mesodermal and neural tissues (Gamer et al., 1999, Dev. Biol. 208:222-32). GDF11 has been shown to be a negative regulator of chondrogenesis and myogenesis in the developing chicken limb (Gamer et al., 2001, Dev. Biol. 229:407-20). GDF11 expression in muscle also suggests its role in regulating muscle growth in a manner similar to GDF8. Furthermore, the expression of GDF11 in the brain suggests that GDF11 may also have activities related to nervous system function. Interestingly, GDF11 was found to inhibit neurogenesis in the olfactory epithelium (Wu et al., 2003, Neuron. 37:197-207).

[0184] In part, the examples of the present disclosure demonstrate that ActRIIB:ALK4 heterodimers are effective in improving diastolic dysfunction as measured by E / e' regardless of age, but that treatment at an earlier age yields more robust results. Furthermore, ActRIIB(K55E)-Fc was shown to improve diastolic dysfunction as measured by E / e'. Compared to WT mice, "db / db-vehicle" mice had no decreased ejection fraction, but increased BNP levels, indicating HFpEF. The data further suggest that, in addition to ActRIIB:ALK4 heterodimers, other ActRII-ALK4 antagonists and ActRII polypeptides and variants may be useful in treating heart failure associated with metabolic complications (e.g., diabetes, obesity).

[0185] In certain aspects, the ActRII-ALK4 antagonist used in accordance with the methods and uses disclosed herein (e.g., treating, preventing, or reducing the progression rate and / or severity of heart failure associated with metabolic complications (e.g., diabetes, obesity), or one or more complications of heart failure) is an ActRII-ALK4 ligand trap polypeptide antagonist, including variants thereof and heterodimers and heteromultimers thereof, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and / or an ActRII-ALK4 small molecule antagonist. ActRII-ALK4 ligand trap polypeptides include TGF-β superfamily-related proteins (including variants thereof) capable of binding to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and / or BMP10). Thus, an ActRII-ALK4 ligand trap generally comprises a polypeptide capable of antagonizing one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and / or BMP10). In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 ligand trap. In some embodiments, an ActRII-ALK4 ligand trap comprises an ActRIIB polypeptide (including variants thereof), and also has homomultimers (e.g., ActRIIB homodimers) and heteromultimers (e.g., ActRIIB-ALK4 or ActRIIB-ALK7 heterodimers). In some embodiments, the ActRII-ALK4 ligand trap comprises an ActRIIA polypeptide (including variants thereof), and includes homomultimers (e.g., ActRIIA homodimers) and heteromultimers (e.g., ActRIIA-ALK4 or ActRIIA-ALK7 heterodimers). In other embodiments, the ActRII-ALK ligand trap comprises a soluble ligand trap protein, including, but not limited to, a follistatin polypeptide and variants thereof.In some embodiments, the ActRII-ALK4 antagonist comprises an ActRII-ALK4 antibody antagonist (an antibody that inhibits one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4, and / or ALK7). In some embodiments, the ActRII-ALK4 antagonist comprises an ActRII-ALK4 small molecule antagonist (e.g., a small molecule that inhibits one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4, and / or ALK7). In some embodiments, the ActRII-ALK4 antagonist comprises an ActRII-ALK4 polynucleotide antagonist (e.g., a nucleotide sequence that inhibits one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and / or ALK7).

[0186] The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and within the specific context in which each term is used. Particular terms are discussed below or elsewhere herein to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which it is used.

[0187] The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

[0188] "Percent (%) sequence identity" with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to those in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid (nucleic acid) sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted along with user documentation to the U.S. Copyright Office, Washington, DC, 20559, where it 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 must be compiled for use on UNIX operating systems, including digital UNIX V 4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0189] "Agonize," in all its grammatical forms, refers to the process of activating a protein and / or gene (e.g., by activating or amplifying gene expression of that protein or by inducing an inactive protein to enter an active state) or increasing the activity of a protein and / or gene.

[0190] "Antagonize," in all its grammatical forms, refers to the process of inhibiting a protein and / or gene (e.g., by inhibiting or reducing gene expression of that protein or by inducing an active protein to enter an inactive state) or reducing the activity of a protein and / or gene.

[0191] The terms "about" and "approximately" as used in connection with numerical values ​​throughout this specification and claims indicate an interval of accuracy familiar and accepted by those skilled in the art. Generally, such an interval of accuracy is ±10%. Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values ​​within an order of magnitude of a given value, preferably within 5-fold or less, and more preferably within 2-fold or less.

[0192] The numerical ranges disclosed herein are inclusive of the numbers defining the range.

[0193] The terms "a" and "an" include plural referents unless the context in which the term is used clearly dictates otherwise. The terms "a" (or "an") and "one or more" and "at least one" can be used interchangeably herein. Furthermore, "and / or," when used herein, should be construed as a specific disclosure of each of two or more specified features or components, regardless of the presence or absence of the others. Thus, herein, the term "and / or," when used in a phrase such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in a phrase such as "A, B, and / or C" is intended to encompass each of 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).

[0194] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0195] 2. ActRII-ALK4 ligand capture antagonists and their variants In certain aspects, the ActRII-ALK4 antagonist used in accordance with the methods and uses disclosed herein (e.g., treating, preventing, or reducing the progression rate and / or severity of heart failure associated with metabolic complications (e.g., diabetes, obesity), or one or more complications of heart failure) is an ActRII-ALK4 ligand trap polypeptide, including variants thereof, and heterodimers and heteromultimers thereof. ActRII-ALK4 ligand trap polypeptides include TGF-β superfamily-related proteins (including variants thereof) that can bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and BMP10). Thus, ActRII-ALK4 ligand traps generally include polypeptides that can antagonize one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and BMP10). For example, in some embodiments, the ActRII-ALK4 ligand trap comprises an ActRII polypeptide, a variant thereof, and homo- and heteromultimers thereof (e.g., homodimers and heterodimers, respectively). As used herein, the term "ActRII" refers to the type II activin receptor family. This family includes activin receptor type IIA (ActRIIA) and activin receptor type IIB (ActRIIB). In some embodiments, the ActRII-ALK4 ligand trap comprises an ActRIIB polypeptide (including a variant thereof), and also has homomultimers (e.g., ActRIIB homodimers) and heteromultimers (e.g., ActRIIB-ALK4 or ActRIIB-ALK7 heterodimers). In some embodiments, the ActRII-ALK4 ligand trap comprises an ActRIIA polypeptide (including variants thereof), and also has homomultimers (e.g., ActRIIA homodimers) and heteromultimers (e.g., ActRIIA-ALK4 or ActRIIA-ALK7 heterodimers).In other embodiments, the ActRII-ALK ligand trap comprises a soluble ligand trap protein, including, but not limited to, a follistatin polypeptide and variants thereof.

[0196] A) ActRIIB polypeptide In certain aspects, the present disclosure relates to ActRII-ALK4 antagonists comprising ActRIIB polypeptides, including fragments, functional variants, and modified forms thereof, and uses thereof (e.g., treating, preventing, or reducing the rate of progression and / or severity of heart failure (HF) or one or more complications of HF). As used herein, the term "ActRIIB" refers to the family of activin receptor type IIB (ActRIIB) proteins of any species, and variant polypeptides derived from such ActRIIB proteins by mutagenesis or other modification (e.g., including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such variant ActRIIB polypeptides are provided throughout this disclosure and in International Patent Application Publication Nos. WO 2006 / 012627, WO 2008 / 097541, WO 2010 / 151426, WO 2011 / 020045, WO 2018 / 009624, and WO 2018 / 067874, which are incorporated by reference in their entireties. References to ActRIIB herein are understood to refer to any one of its currently identified forms. Members of the ActRIIB family are generally all transmembrane polypeptides, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase specificity. The amino acid sequence of a human ActRIIB precursor polypeptide is shown in Figure 2 (SEQ ID NO: 2) and below. Preferably, the ActRIIB polypeptides used in accordance with the methods of the present disclosure are soluble. As used herein, the term "soluble ActRIIB polypeptide" includes any naturally occurring extracellular domain of an ActRIIB polypeptide, as well as any variant thereof (including mutants, fragments, and peptidomimetic forms) that retains useful activity. For example, the extracellular domain of an ActRIIB polypeptide binds to a ligand and is generally soluble. Examples of soluble ActRIIB polypeptides include the ActRIIB extracellular domain shown in Figure 3 (SEQ ID NO: 1) and SEQ ID NO: 53.This truncated ActRIIB extracellular domain (SEQ ID NO: 53) is designated ActRIIB(25-131) based on the numbering of SEQ ID NO: 2. Other examples of soluble ActRIIB polypeptides include a signal sequence in addition to the extracellular domain of the ActRIIB polypeptide (see Example 4). The signal sequence can be the native signal sequence of ActRIIB or a signal sequence from another polypeptide, such as the tissue plasminogen activator (TPA) signal sequence or the honeycomb melittin signal sequence. In some embodiments, the ActRIIB polypeptide inhibits one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) (e.g., Smad signaling). In some embodiments, the ActRIIB polypeptide binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Various exemplary methods and assays for determining the ability of an ActRIIB polypeptide to bind to and / or inhibit the activity of one or more ActRII-ALK4 ligands are disclosed herein or are otherwise known in the art and can be readily used to determine whether an ActRIIB polypeptide has the desired binding and / or antagonistic activity. The amino acid numbering of all ActRIIB-related polypeptides described herein is based on the numbering of the human ActRIIB precursor protein sequence (SEQ ID NO: 2) provided below, unless otherwise specified.

[0197] The human ActRIIB precursor protein sequence is as follows:

[0198] [ka]

[0199] The signal peptide is Single underscore The extracellular domain is shown in bold and potential endogenous N-linked glycosylation sites are double underline is shown.

[0200] The processed (mature) extracellular ActRIIB polypeptide sequence is as follows:

[0201] TIFF2024511316000003.tif21141

[0202] In some embodiments, the protein can be produced with an "SGR..." sequence at the N-terminus. The C-terminal "tail" of the extracellular domain is Single underscore The "tail" deleted sequence (Δ15 sequence) is as follows:

[0203] [ka]

[0204] Forms of ActRIIB with an alanine (A64) at position 64 of SEQ ID NO:2 have also been reported in the literature. See, e.g., Hilden et al. (1994) Blood, 83(8):2163-2170. Applicants have confirmed that an ActRIIB-Fc fusion protein containing the extracellular domain of ActRIIB with an A64 substitution has relatively low affinity for activin and GDF11. In contrast, the same ActRIIB-Fc fusion protein with an arginine (R64) at position 64 has affinity for activin and GDF11 in the low nanomolar to high picomolar range. Therefore, the sequence with R64 is used as the "wild-type" reference sequence for human ActRIIB in the present disclosure.

[0205] The ActRIIB precursor protein sequence with an alanine at position 64 is as follows:

[0206] [ka]

[0207] Signal peptide Single underscoreand the extracellular domain is shown in bold.

[0208] The processed (mature) extracellular ActRIIB polypeptide sequence of the alternative A64 form is as follows:

[0209] [ka]

[0210] In some embodiments, the protein can be produced with an "SGR..." sequence at the N-terminus. The C-terminal "tail" of the extracellular domain is Single underscore The polypeptide sequence of an alternative A64 form with the "tail" deleted (the Δ15 sequence) is as follows:

[0211] [ka]

[0212] The nucleic acid sequence encoding the human ActRIIB precursor protein is shown below (SEQ ID NO:4) and represents nucleotides 25-1560 of GenBank reference sequence NM_001106.3, which encodes amino acids 1-513 of the ActRIIB precursor. The nucleotide sequence shown encodes a polypeptide with an arginine at position 64, and can be modified to encode a polypeptide with an alanine instead. The signal sequence is underlined.

[0213] [ka] TIFF2024511316000009.tif145141

[0214] The nucleic acid sequence encoding the processed extracellular human ActRIIB polypeptide is as follows (SEQ ID NO: 3): The nucleotide sequence shown encodes a polypeptide having an arginine at position 64, and can be modified to encode a polypeptide having an alanine instead (see Figure 5, SEQ ID NO: 3).

[0215] [ka]

[0216] B) Variant ActRIIB polypeptides In certain specific embodiments, the present disclosure contemplates making mutations in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIB polypeptide such that the variant (or mutant) ActRIIB polypeptide has altered ligand-binding activity (e.g., binding affinity or binding selectivity). In certain cases, such variant ActRIIB polypeptides have altered (increased or decreased) binding affinity for a specific ligand. In other cases, variant ActRIIB polypeptides have altered binding selectivity for their ligands. For example, the present disclosure provides numerous mutant ActRIIB polypeptides that have reduced binding affinity for BMP9, but retain binding affinity for one or more of activin A, activin B, GDF8, GDF11, and BMP10, compared to unmodified ActRIIB polypeptides. In some cases, variant ActRIIB polypeptides have similar or the same biological activity as their corresponding wild-type ActRIIB polypeptides. For example, a variant ActRIIB polypeptide of the present disclosure may bind to and inhibit the function of an ActRIIB ligand (e.g., activin A, activin B, GDF8, GDF11, or BMP10). In some embodiments, a variant ActRIIB polypeptide of the present disclosure treats, prevents, or reduces the rate of progression and / or severity of heart failure or one or more complications of heart failure. Examples of ActRIIB polypeptides include human ActRIIB precursor polypeptide (SEQ ID NO: 2 and SEQ ID NO: 387) and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 1, 5, 6, 12, 276, 278, 279, 332, 333, 335, 336, 338, 339, 341, 342, 344, 345, 347, 348, 350, 351, 353, 354, 356, 357, 385, 386, 387, 388, 389, 396, 398, 402, 403, 406, 408, and 409). In some embodiments, the variant ActRIIB polypeptide is a member of a homomultimer (e.g., a homodimer). In some embodiments, the variant ActRIIB polypeptide is a member of a heteromultimer (e.g., a heterodimer).In some embodiments, any of the variant ActRIIB polypeptides may be combined (e.g., heteromultimerized and / or fused) with any of the polypeptides disclosed herein.

[0217] ActRIIB is well conserved across almost all vertebrates, with a large stretch of the extracellular domain being completely conserved. See, for example, Figure 6. Many of the ligands that bind to ActRIIB are also highly conserved. Thus, comparison of ActRIIB sequences from various vertebrates provides insight into residues that may vary. Thus, an active human ActRIIB variant may contain one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIB, or may contain residues similar to those in the sequence of a human or other vertebrate.

[0218] The present disclosure identifies functionally active portions and variants of ActRIIB. Applicant has previously confirmed that an Fc fusion polypeptide having the sequence disclosed by Hilden et al. (Blood. 1994 Apr 15;83(8):2163-70), which has an alanine at position corresponding to amino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity for activin and GDF11. In contrast, the same Fc fusion polypeptide having an arginine at position 64 (R64) has affinity for activin and GDF-11 in the low nanomolar to high picomolar range. Therefore, the sequence having R64 (SEQ ID NO: 2) is used as the wild-type reference sequence for human ActRIIB in the present disclosure, and the numbering of the variants described herein is based on the numbering of SEQ ID NO: 2. Furthermore, one skilled in the art can generate any of the ActRIIB variants described herein in an A64 background.

[0219] The processed extracellular ActRIIB polypeptide sequence is set forth in SEQ ID NO: 1 (see, e.g., Figure 3). In some embodiments, the processed ActRIIB polypeptide may be produced with an "SGR..." sequence at the N-terminus. In some embodiments, the processed ActRIIB polypeptide may be produced with a "GRG..." sequence at the N-terminus. For example, some constructs are expected to lack an N-terminal serine when expressed with a TPA leader. Thus, the mature ActRIIB sequence described herein may begin with either an N-terminal serine or an N-terminal glycine (lacking an N-terminal serine).

[0220] Attisano et al. (Cell. 1992 Jan. 10;68(1):97-108) showed that deletion of the C-terminal proline knot in the extracellular domain of ActRIIB reduces the receptor's affinity for activin. Data disclosed in WO 2008097541 show that an ActRIIB-Fc fusion polypeptide containing amino acids 20 to 119 of SEQ ID NO: 2, "ActRIIB(20-119)-Fc," exhibits reduced binding to GDF11 and activin compared with ActRIIB(20-134)-Fc, which contains the proline knot region and the entire adjacent membrane domain. However, the ActRIIB(20-129)-Fc polypeptide retains similar, though somewhat reduced, activity compared to the wild-type, despite the disruption of the proline knot region. Thus, ActRIIB extracellular domains ending at amino acids 134, 133, 132, 131, 130, and 129 are all expected to be active, but constructs ending at 134 or 133 may be the most active. Similarly, mutations at any of residues 129-134 are not expected to alter ligand binding affinity by a large margin. In support of this, mutations at P129 and P130 do not substantially reduce ligand binding. Thus, ActRIIB-Fc fusion polypeptides can terminate as early as amino acid 109 (the final cysteine), but forms ending at 109-119 are expected to have reduced ligand binding. Amino acid 119 is less conserved and therefore easily altered or truncated. Forms ending at 128 or later retain ligand binding activity. Forms ending at 119 or 127 have intermediate binding capacity. Depending on the clinical or experimental setting, it may be desirable to use one of these forms.

[0221] At the N-terminus of ActRIIB, polypeptides beginning at or before amino acid 29 are predicted to retain ligand-binding activity. Amino acid 29 represents an initial cysteine. Mutation of alanine to asparagine at position 24 introduces an N-linked glycosylation sequence without substantially affecting ligand binding. This confirms that mutations in the region between the signal cleavage peptide and the cysteine ​​bridge region corresponding to amino acids 20-29 are well tolerated. In particular, constructs beginning at positions 20, 21, 22, 23, and 24 are predicted to retain activity, as are constructs beginning at positions 25, 26, 27, 28, and 29. Data presented in WO2008097541 surprisingly demonstrate that constructs beginning at positions 22, 23, 24, or 25 have the highest activity.

[0222] In summary, the active portion of ActRIIB comprises amino acids 29 to 109 of SEQ ID NO: 2, and the construct can, for example, begin at a residue corresponding to amino acids 20 to 29 and end at a position corresponding to amino acids 109 to 134. Other examples include constructs beginning at positions 20 to 29 or 21 to 29 and ending at positions 119 to 134, 119 to 133, 129 to 134, or 129 to 133. Other examples include constructs beginning at positions 20 to 24 (or 21 to 24 or 22 to 25) and ending at positions 109 to 134 (or 109 to 133), 119 to 134 (or 119 to 133), or 129 to 134 (or 129 to 133). Variants within these ranges are also contemplated, particularly variants having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding portion of SEQ ID NO:1.

[0223] The variations described herein can be combined in various ways. In some embodiments, ActRIIB variants contain no more than 1, 2, 5, 6, 7, 8, 9, 10, or 15 conservative amino acid changes within the ligand-binding pocket, and may contain zero, one, or more non-conservative changes at positions 40, 53, 55, 74, 79, and / or 82 within the ligand-binding pocket. Sites outside the binding pocket where variability may be particularly well tolerated include the amino and carboxy termini of the extracellular domain (as described above), as well as positions 42-46 and 65-73 (with respect to SEQ ID NO: 2). An asparagine-to-alanine change at position 65 (N65A) does not appear to reduce ligand binding in the R64 background [U.S. Pat. No. 7,842,663]. This change likely eliminates glycosylation at N65 in the A64 background, demonstrating that significant variation in this region is likely to be tolerated. While the R64A change is poorly tolerated, R64K is well tolerated, and therefore, another basic residue, such as H, may be tolerated at position 64 [U.S. Pat. No. 7,842,663]. Furthermore, results of mutagenesis programs described in the art indicate that there are amino acid positions in ActRIIB that are often beneficial to conserve. With respect to SEQ ID NO:2, these include position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, particularly tryptophan), position 37 (acidic, particularly aspartic acid or glutamic acid), position 56 (basic amino acid), and position 60 (hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus, the present disclosure provides a framework of amino acids that may be conserved in ActRIIB polypeptides. Other positions that may be desirable to conserve are: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), and position 98 (polar or charged, particularly E, D, R, or K), all with respect to SEQ ID NO:2.

[0224] The addition of additional N-linked glycosylation sites (NXS / T) to the ActRIIB extracellular domain has previously been demonstrated to be well tolerated (see, e.g., U.S. Pat. No. 7,842,663). Therefore, NXS / T sequences can generally be introduced into the ActRIIB polypeptides of the present disclosure at positions outside the ligand-binding pocket defined in FIG. 1 . Particularly suitable sites for the introduction of non-endogenous NXS / T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134, or 129-134 (with respect to SEQ ID NO: 2). NXS / T sequences may also be introduced into the linker between the ActRIIB sequence and the Fc domain or other fusion component, as well as, optionally, into the fusion component itself. Such sites can be introduced with minimal effort by introducing an N at the correct position relative to an existing S or T, or by introducing an S or T at the position corresponding to an existing N. Thus, desirable changes that would create N-linked glycosylation sites are A24N, R64N, S67N (possibly in combination with an N65A change), E105N, R112N, G120N, E123N, P129N, A132N, R112S, and R112T (with respect to SEQ ID NO: 2). Any S predicted to be glycosylated can be changed to T without creating an immunogenic site due to the protection afforded by glycosylation. Similarly, any T predicted to be glycosylated can be changed to S. Thus, the changes S67T and S44T (with respect to SEQ ID NO: 2) are contemplated. Similarly, in the A24N variant, an S26T modification can be used. Thus, the ActRIIB polypeptides of the present disclosure can be variants having one or more additional non-endogenous N-linked glycosylation consensus sequences as described above.

[0225] In certain embodiments, the variant ActRIIB polypeptide has an amino acid sequence at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, and 53. In certain cases, the variant ActRIIB polypeptide has an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, and 53. In certain cases, the variant ActRIIB polypeptide has an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In certain cases, the variant ActRIIB polypeptide has an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In certain cases, the variant ActRIIB polypeptide has an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53.

[0226] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the disclosure is selected from the group consisting of SEQ ID NOs: 1, 2, 5, 6, 12, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 50, 51, 52, 53, 276, 278, 279, 332, 333, 335, 336, 338, 339, 341, 342, 344, 345, 347, 348, 350, 351, 353, 354, 356, 357, 385, 386, 387, 388, 389, 396, 398, 402, 403, 406, 408, and 409. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 1. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 1 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 2. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 2 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 5. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 5 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 6. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 6 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 12. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 12 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 31.

[0227] An ActRIIB-Fc fusion protein comprising SEQ ID NO: 31 may be provided with the lysine removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 33. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 33 may be provided with the lysine removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 34. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 34 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 36. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 36 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 37.An ActRIIB-Fc fusion protein comprising SEQ ID NO: 37 may be provided with the lysine removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 39. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 39 may be provided with the lysine removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 40. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 40 may be provided with lysines removed from the C-terminus.

[0228] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 42. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 42 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 43. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 43 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 45. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 45 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 46. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 46 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 48. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 48 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 49. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 49 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 50. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 50 may be provided with lysines removed from the C-terminus.

[0229] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 51. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 51 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 52. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 52 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 53. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 53 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 276. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 276 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 278. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 278 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 279. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 279 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 332. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 332 may be provided with lysines removed from the C-terminus.

[0230] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 333. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 333 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 335. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 335 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 336. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 336 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 338. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 338 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 339. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 339 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 341. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 341 may be provided with lysines removed from the C-terminus.

[0231] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 342. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 342 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 344. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 344 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 345. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 345 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 347. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 347 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 348. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 348 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 350. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 350 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 351. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 351 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 353. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 353 may be provided with lysines removed from the C-terminus.

[0232] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 354. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 354 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 356. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 356 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 357. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 357 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 385. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 385 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 386. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 386 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 387. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 387 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 388. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 388 may be provided with lysines removed from the C-terminus.

[0233] In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 389. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 389 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 396. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 396 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 398. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 398 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 402. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 402 may be provided with lysines removed from the C-terminus.In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 403. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 403 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 406. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 406 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 408. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 408 may be provided with lysines removed from the C-terminus. In some embodiments, a variant ActRIIB polypeptide or variant ActRIIB-Fc fusion polypeptide of the present disclosure comprises, consists of, or consists essentially 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 the amino acid sequence of SEQ ID NO: 409. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 409 may be provided with lysines removed from the C-terminus.

[0234] In certain aspects, the present disclosure provides an amino acid sequence that is at least 70%, 75%, or more similar to an amino acid sequence beginning with any one of amino acids 20 to 29 of SEQ ID NO:2 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) and ending with any one of amino acids 109 to 134 of SEQ ID NO:2 (e.g., amino acid residues 109, 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 134). , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, wherein the polypeptide comprises one or more amino acid substitutions at positions selected from the group consisting of K55, F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82, and a heteromultimeric complex comprising one or more such variant ActRIIB polypeptides. In certain aspects, the present disclosure provides a method for the preparation of a nucleotide sequence beginning with any one of amino acids 20-29 of SEQ ID NO:2 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) and ending with any one of amino acids 109-134 of SEQ ID NO:2 (e.g., amino acid residues 109, 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 134). and a variant ActRIIB polypeptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence ending in SEQ ID NO: 2, wherein the polypeptide contains one or more amino acid substitutions at positions in SEQ ID NO: 2, except that the amino acid at position 79 in SEQ ID NO: 2 is leucine, as well as heteromultimeric complexes comprising one or more such variant ActRIIB polypeptides.In some embodiments, the variant ActRIIB polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-131 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 20-134 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the variant ActRIIB polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the variant ActRIIB polypeptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to A24 of SEQ ID NO: 2. For example, in some embodiments, the substitution is A24N. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to S26 of SEQ ID NO: 2. For example, in some embodiments, the substitution is S26T. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to N35 of SEQ ID NO: 2. For example, in some embodiments, the substitution is N35E.In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E37 in SEQ ID NO:2. For example, in some embodiments, the substitution is E37A. In some embodiments, the substitution is E37D. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to L38 in SEQ ID NO:2. For example, in some embodiments, the substitution is L38N. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to R40 in SEQ ID NO:2. For example, in some embodiments, the substitution is R40A. In some embodiments, the substitution is R40K. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to S44 in SEQ ID NO:2. For example, in some embodiments, the substitution is S44T. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to L46 in SEQ ID NO:2. For example, in some embodiments, the substitution is L46A. For example, in some embodiments, the substitution is L46I. For example, in some embodiments, the substitution is L46F. For example, in some embodiments, the substitution is L46V. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E50 in SEQ ID NO:2. For example, in some embodiments, the substitution is E50K. In some embodiments, the substitution is E50L. In some embodiments, the substitution is E50P. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E52 of SEQ ID NO:2. For example, in some embodiments, the substitution is E52A. In some embodiments, the substitution is E52D. In some embodiments, the substitution is E52G. In some embodiments, the substitution is E52H. In some embodiments, the substitution is E52K. In some embodiments, the substitution is E52N. In some embodiments, the substitution is E52P. In some embodiments, the substitution is E52R. In some embodiments, the substitution is E52S. In some embodiments, the substitution is E52T. In some embodiments, the substitution is E52Y. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to Q53 of SEQ ID NO:2.For example, in some embodiments, the substitution is Q53R. For example, in some embodiments, the substitution is Q53K. For example, in some embodiments, the substitution is Q53N. For example, in some embodiments, the substitution is Q53H. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to D54 of SEQ ID NO:2. For example, in some embodiments, the substitution is D54A. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to K55 of SEQ ID NO:2. For example, in some embodiments, the substitution is K55A. In some embodiments, the substitution is K55E. In some embodiments, the substitution is K55D. In some embodiments, the substitution is K55R. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to R56 of SEQ ID NO:2. For example, in some embodiments, the substitution is R56A. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to L57 of SEQ ID NO:2. For example, in some embodiments, the substitution is L57R. In some embodiments, the substitution is L57E. In some embodiments, the substitution is L57I. In some embodiments, the substitution is L57T. In some embodiments, the substitution is L57V. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to Y60 in SEQ ID NO:2. For example, in some embodiments, the substitution is Y60F. In some embodiments, the substitution is Y60D. In some embodiments, the substitution is Y60K. In some embodiments, the substitution is Y60P. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to R64 in SEQ ID NO:2. For example, in some embodiments, the substitution is R64K. In some embodiments, the substitution is R64N. In some embodiments, the substitution is R64A. In some embodiments, the substitution is R64H. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to N65 in SEQ ID NO:2. For example, in some embodiments, the substitution is N65A.In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to S67 in SEQ ID NO:2. For example, in some embodiments, the substitution is S67N. In some embodiments, the substitution is S67T. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to G68 in SEQ ID NO:2. For example, in some embodiments, the substitution is G68R. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to K74 in SEQ ID NO:2. For example, in some embodiments, the substitution is K74A. In some embodiments, the substitution is K74E. In some embodiments, the substitution is K74F. In some embodiments, the substitution is K74I. In some embodiments, the substitution is K74Y. In some embodiments, the substitution is K74R. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to W78 in SEQ ID NO:2. For example, in some embodiments, the substitution is W78A. In some embodiments, the substitution is W78Y. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to L79 in SEQ ID NO:2. For example, in some embodiments, the substitution is L79D. In some embodiments, the substitution does not include an acidic amino acid at a position corresponding to L79 of SEQ ID NO:2. In some embodiments, the substitution is not at position L79 of SEQ ID NO:2. In some embodiments, position L79 of SEQ ID NO:2 is not substituted. In some embodiments, the substitution does not include an aspartic acid (D) at a position corresponding to L79 of SEQ ID NO:2. In some embodiments, the substitution is L79A. In some embodiments, the substitution is L79E. In some embodiments, the substitution is L79F. In some embodiments, the substitution is L79H. In some embodiments, the substitution is L79K. In some embodiments, the substitution is L79P. In some embodiments, the substitution is L79R. In some embodiments, the substitution is L79S. In some embodiments, the substitution is L79T. In some embodiments, the substitution is L79W. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to D80 of SEQ ID NO:2.For example, in some embodiments, the substitution is D80A. In some embodiments, the substitution is D80F. In some embodiments, the substitution is D80K. In some embodiments, the substitution is D80G. In some embodiments, the substitution is D80M. In some embodiments, the substitution is D80I. In some embodiments, the substitution is D80N. In some embodiments, the substitution is D80R. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to F82 in SEQ ID NO:2. For example, in some embodiments, the substitution is F82I. In some embodiments, the substitution is F82K. In some embodiments, the substitution is F82A. In some embodiments, the substitution is F82W. In some embodiments, the substitution is F82D. In some embodiments, the substitution is F82Y. In some embodiments, the substitution is F82E. In some embodiments, the substitution is F82L. In some embodiments, the substitution is F82T. In some embodiments, the substitution is F82S. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to N83 in SEQ ID NO:2. For example, in some embodiments, the substitution is N83A. In some embodiments, the substitution is N83R. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to T93 in SEQ ID NO:2. For example, in some embodiments, the substitution is T93D. In some embodiments, the substitution is T93E. In some embodiments, the substitution is T93H. In some embodiments, the substitution is T93G. In some embodiments, the substitution is T93K. In some embodiments, the substitution is T93P. In some embodiments, the substitution is T93R. In some embodiments, the substitution is T93S. In some embodiments, the substitution is T93Y. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E94 in SEQ ID NO:2. For example, in some embodiments, the substitution is E94K. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to Q98 in SEQ ID NO:2. For example, in some embodiments, the substitution is Q98D. In some embodiments, the substitution is Q98E. In some embodiments, the substitution is Q98K. In some embodiments, the substitution is Q98R. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to V99 in SEQ ID NO:2. For example, in some embodiments, the substitution is V99E. In some embodiments, the substitution is V99G. In some embodiments, the substitution is V99K. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E105 in SEQ ID NO:2. For example, in some embodiments, the substitution is E105N.In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E106 of SEQ ID NO:2. For example, in some embodiments, the substitution is E106N. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to F108 of SEQ ID NO:2. For example, in some embodiments, the substitution is F108I. In some embodiments, the substitution is F108L. In some embodiments, the substitution is F108V. In some embodiments, the substitution is F108Y. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E111 of SEQ ID NO:2. For example, in some embodiments, the substitution is E111K. In some embodiments, the substitution is E111D. In some embodiments, the substitution is E111R. In some embodiments, the substitution is E111H. In some embodiments, the substitution is E111Q. In some embodiments, the substitution is E111N. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to R112 of SEQ ID NO:2. For example, in some embodiments, the substitution is R112H. In some embodiments, the substitution is R112K. In some embodiments, the substitution is R112N. In some embodiments, the substitution is R112S. In some embodiments, the substitution is R112T. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to A119 in SEQ ID NO:2. For example, in some embodiments, the substitution is A119P. In some embodiments, the substitution is A119V. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to G120 in SEQ ID NO:2. For example, in some embodiments, the substitution is G120N. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to E123 in SEQ ID NO:2. For example, in some embodiments, the substitution is E123N. In some embodiments, the polypeptide comprises an amino acid substitution at an amino acid position corresponding to P129 in SEQ ID NO:2. For example, in some embodiments, the substitution is P129S. In some embodiments, the substitution is P129N.In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to P130 of SEQ ID NO: 2. For example, in some embodiments, the substitution is P130A. In some embodiments, the substitution is P130R. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to A132 of SEQ ID NO: 2. For example, in some embodiments, the substitution is A132N.

[0235] In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises a substitution at a position of SEQ ID NO:2 selected from the group consisting of A24, E37, R40, D54, K55, R56, R64, K74, W78, L79, D80, F82, P129, and P130. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position A24 relative to SEQ ID NO:2. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position E37 relative to SEQ ID NO:2. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position R40 relative to SEQ ID NO:2. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position D54 relative to SEQ ID NO:2. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position K55 relative to SEQ ID NO:2. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position R56 relative to SEQ ID NO:2. In some embodiments, a variant ActRIIB polypeptide comprises a substitution at position R64 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position K74 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position W78 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position L79 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position D80 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position F82 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position P129 relative to SEQ ID NO:2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position P130 relative to SEQ ID NO:2.

[0236] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide 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 the amino acid sequence of SEQ ID NO: 31. In some embodiments, the variant ActRIIB polypeptide comprises an alanine at a position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 31 may be provided with a lysine removed from the C-terminus.

[0237] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the variant ActRIIB polypeptide comprises an alanine at a position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 33 may be provided with a lysine removed from the C-terminus.

[0238] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide 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 the amino acid sequence of SEQ ID NO: 34. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at a position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 34 may be provided with a lysine removed from the C-terminus.

[0239] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at a position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 36 may be provided with a lysine removed from the C-terminus.

[0240] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 37 may be provided with a lysine removed from the C-terminus.

[0241] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 39 may be provided with a lysine removed from the C-terminus.

[0242] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 40 may be provided with the lysine removed from the C-terminus.

[0243] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide 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 the amino acid sequence of SEQ ID NO: 42. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 42 may be provided with the lysine removed from the C-terminus.

[0244] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 43 may be provided with a lysine removed from the C-terminus.

[0245] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 45 may be provided with a lysine removed from the C-terminus.

[0246] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 336. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 336 may be provided with a lysine removed from the C-terminus.

[0247] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 338. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 338 may be provided with a lysine removed from the C-terminus.

[0248] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 342. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 342 may be provided with a lysine removed from the C-terminus.

[0249] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 344. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 344 may be provided with a lysine removed from the C-terminus.

[0250] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 348. In some embodiments, the variant ActRIIB polypeptide comprises a leucine at a position corresponding to E50 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 348 may be provided with a lysine removed from the C-terminus.

[0251] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 350. In some embodiments, the variant ActRIIB polypeptide comprises a leucine at a position corresponding to E50 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 350 may be provided with a lysine removed from the C-terminus.

[0252] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 354. In some embodiments, the variant ActRIIB polypeptide comprises a glycine at a position corresponding to V99 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 354 may be provided with a lysine removed from the C-terminus.

[0253] In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 356. In some embodiments, the variant ActRIIB polypeptide comprises a glycine at a position corresponding to V99 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 356 may be provided with a lysine removed from the C-terminus.

[0254] In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises two of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises three of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises four of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises five of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises six of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises seven of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises eight of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises nine of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises ten of any of the amino acid substitutions disclosed herein.

[0255] In certain aspects, the present disclosure relates to variant ActRIIB polypeptides comprising two or more amino acid substitutions compared to the reference amino acid sequence of SEQ ID NO: 2. For example, in some embodiments, a variant ActRIIB polypeptide comprises an A24N substitution and a K74A substitution. In some embodiments, a variant ActRIIB polypeptide comprises an L79P substitution and a K74A substitution. In some embodiments, a variant ActRIIB polypeptide comprises a P129S substitution and a P130A substitution. In some embodiments, a variant ActRIIB polypeptide comprises an L38N substitution and an L79R substitution. In some embodiments, a variant ActRIIB polypeptide comprises an F82I substitution and an N83R substitution. In some embodiments, a variant ActRIIB polypeptide comprises an F82K substitution and an N83R substitution. In some embodiments, a variant ActRIIB polypeptide comprises an F82T substitution and an N83R substitution. In some embodiments, a variant ActRIIB polypeptide comprises an L79H substitution and an F82K substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L79H substitution and an F82I substitution. In some embodiments, the variant ActRIIB polypeptide comprises an F82D substitution and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an F82E substitution and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L79F substitution and an F82D substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L79F substitution and an F82T substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52D substitution and an F82D substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52D substitution and an F82T substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57R substitution and an F82D substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57R substitution and an F82T substitution. In some embodiments, the variant ActRIIB polypeptide comprises an F82I substitution and an E94K substitution.In some embodiments, the variant ActRIIB polypeptide comprises an F82S substitution and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57R substitution and an F82S substitution. In some embodiments, the variant ActRIIB polypeptide comprises a K74A substitution and an L79P substitution. In some embodiments, the variant ActRIIB polypeptide comprises a K55A substitution and an F82I substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L79K substitution and an F82K substitution. In some embodiments, the variant ActRIIB polypeptide comprises an F82W substitution and an N83A substitution.

[0256] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 276. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2 and an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 276 may be provided with a lysine removed from the C-terminus.

[0257] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 278. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2 and an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 278 may be provided with a lysine removed from the C-terminus.

[0258] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 279. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2 and an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 279 may be provided with a lysine removed from the C-terminus.

[0259] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 332. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2 and an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 332 may be provided with a lysine removed from the C-terminus.

[0260] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 333. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at a position corresponding to F82 of SEQ ID NO: 2 and an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 333 may be provided with a lysine removed from the C-terminus.

[0261] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 335. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at a position corresponding to F82 of SEQ ID NO: 2 and an arginine at a position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 335 may be provided with a lysine removed from the C-terminus.

[0262] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 339. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2 and an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 339 may be provided with a lysine removed from the C-terminus.

[0263] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 341. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2 and an isoleucine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 341 may be provided with a lysine removed from the C-terminus.

[0264] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 345. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2 and a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 345 may be provided with a lysine removed from the C-terminus.

[0265] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 347. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at a position corresponding to L79 of SEQ ID NO: 2 and a lysine at a position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 347 may be provided with a lysine removed from the C-terminus.

[0266] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 351. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at a position corresponding to L38 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at a position corresponding to L38 of SEQ ID NO: 2 and an arginine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 351 may be provided with a lysine removed from the C-terminus.

[0267] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 353. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at a position corresponding to L38 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at a position corresponding to L38 of SEQ ID NO: 2 and an arginine at a position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 353 may be provided with a lysine removed from the C-terminus.

[0268] In certain aspects, the present disclosure relates to a variant ActRIIB polypeptide comprising three or more amino acid substitutions compared to the reference amino acid sequence of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, an F82S substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, a W78Y substitution, and an F82Y substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52D substitution, an F82D substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52Y substitution, an F82D substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52D substitution, an F82E substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52D substitution, an F82T substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52N substitution, an F82I substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52N substitution, an F82Y substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E50L substitution, an F82D substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57I substitution, an F82D substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57V substitution, an F82D substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57R substitution, an F82D substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57E substitution, an F82E substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57R substitution, an F82E substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57I substitution, an F82E substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57R substitution, an F82L substitution, and an N83R substitution.In some embodiments, the variant ActRIIB polypeptide comprises an L57T substitution, an F82Y substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an L57V substitution, an F82Y substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide may comprise at least two of the amino acid substitutions set forth in any of the variant ActRIIB polypeptides above.

[0269] In certain aspects, the present disclosure relates to variant ActRIIB polypeptides comprising four or more amino acid substitutions compared to the reference amino acid sequence of SEQ ID NO: 2. For example, in some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, an L79E substitution, an F82Y substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, an L79E substitution, an F82T substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, an L79T substitution, an F82T substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52N substitution, a G68R substitution, an F82Y substitution, and an N83R substitution. In some embodiments, the variant ActRIIB polypeptide may comprise at least two of the amino acid substitutions set forth in any of the variant ActRIIB polypeptides above. In some embodiments, the variant ActRIIB polypeptide may include at least three of the amino acid substitutions set forth in any of the above variant ActRIIB polypeptides.

[0270] C) ActRIIA polypeptide In certain embodiments, the present disclosure relates to ActRII-ALK4 antagonists, including ActRIIA polypeptides, including fragments, functional variants, and modified forms thereof, and uses thereof (e.g., treating, preventing, or reducing the rate of progression and / or severity of heart failure (HF) or one or more complications of HF). As used herein, the term "ActRIIA" refers to any species of the family of activin receptor type IIA (ActRIIA) proteins, and variant polypeptides derived from such ActRIIA proteins by mutagenesis or other modifications (e.g., mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such variant ActRIIA polypeptides are provided throughout this disclosure and in International Patent Application Publication Nos. 2006 / 012627 and 2007 / 062188, which are incorporated herein by reference in their entireties. References herein to ActRIIA are understood to refer to any one of the currently identified forms. ActRIIA family members are generally transmembrane proteins composed of a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity. Preferably, the ActRIIA polypeptide used in accordance with the methods of the present disclosure is soluble (e.g., the extracellular domain of ActRIIA). In some embodiments, the ActRIIA polypeptide inhibits one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) (e.g., Smad signaling). In some embodiments, the ActRIIA polypeptide binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10).Various exemplary methods and assays for determining the ability of an ActRIIA polypeptide to bind to and / or inhibit the activity of one or more ActRII-ALK4 ligands are disclosed herein or are otherwise known in the art and can be readily used to determine whether an ActRIIA polypeptide has the desired binding and / or antagonistic activity. The amino acid numbering of all ActRIIA-related polypeptides described herein is based on the numbering of the human ActRIIA precursor protein sequence (SEQ ID NO: 366) provided below, unless otherwise specified.

[0271] The canonical human ActRIIA precursor protein sequence is as follows:

[0272] [ka]

[0273] The signal peptide is Single underscore The extracellular domain is shown in bold and potential endogenous N-linked glycosylation sites are double underline is shown.

[0274] The processed (mature) extracellular human ActRIIA polypeptide sequence is as follows:

[0275] [ka]

[0276] The C-terminal "tail" of the extracellular domain is Single underscore The "tail" deleted sequence (Δ15 sequence) is as follows:

[0277] [ka]

[0278] The nucleic acid sequence encoding the human ActRIIA precursor protein is shown below (SEQ ID NO: 369), corresponding to nucleotides 159-1700 of GenBank reference sequence NM_001616.4. underline is attached.

[0279] [ka]

[0280] The nucleic acid sequence encoding the processed soluble (extracellular) human ActRIIA polypeptide is as follows:

[0281] [ka]

[0282] ActRIIA is well conserved among vertebrates, with large stretches of the extracellular domain being completely conserved. For example, Figure 10 shows a multiple sequence alignment of the human ActRIIA extracellular domain (SEQ ID NO: 367) compared with various ActRIIA orthologs (SEQ ID NOs: 371-377). Many of the ligands that bind to ActRIIA are also highly conserved. Therefore, from these alignments, it is possible to predict important amino acid positions within the ligand-binding domain that are important for normal ActRIIA-ligand binding activity, as well as amino acid positions that are likely to tolerate substitutions without significantly altering normal ActRIIA-ligand binding activity. Thus, active human ActRIIA variant polypeptides useful according to the methods of the present disclosure may contain one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIA, or may contain residues similar to those in the sequence of a human or other vertebrate.

[0283] Although not meant to be limiting, the following example illustrates this approach to defining active ActRIIA variants. As shown in Figure 10, F13 of the human extracellular domain is Y in Ovis aries (SEQ ID NO: 371), Gallus gallus (SEQ ID NO: 374), Bos taurus (SEQ ID NO: 375), Tyto alba (SEQ ID NO: 376), and Myotis davidii (SEQ ID NO: 377) ActRIIA, indicating that aromatic residues, including F, W, and Y, are tolerated at this position. Q24 in the human extracellular domain is R in Bos taurus ActRIIA, indicating that charged residues, including D, R, K, H, and E, are tolerated at this position. S95 in the human extracellular domain is F in Gallus gallus and F in Tyto alba ActRIIA, indicating that this site can tolerate a 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 Ovis aries ActRIIA, indicating that acidic residues, including D and E, are tolerated at this position in the human extracellular domain. P29 in the human extracellular domain is relatively less conserved and appears as S in Ovis aries ActRIIA and L in Myotis davidii ActRIIA, indicating that essentially any amino acid should be tolerated at this position.

[0284] Furthermore, as noted above, ActRII proteins have been characterized in the art with respect to structural and functional properties, 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) Proc Natl Acad Sci USA 103(20:7643-7648; Thompson et al. (2003) The EMBO Journal 22(7):1555-1566; and U.S. Patent Nos. 7,709,605, 7,612,041, and 7,842,663]. In addition to the teachings herein, these references provide ample guidance on how to generate ActRII variants that retain one or more desired activities (e.g., ligand binding activity).

[0285] For example, a defining structural motif known as the three-finger toxin fold, which is important for ligand binding by type I and type II receptors, is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Thus, the core ligand-binding domain of human ActRIIA, defined by the outermost of these conserved cysteines, corresponds to positions 30-110 of SEQ ID NO: 366 (ActRIIA precursor). Thus, the structurally unordered amino acids adjacent to these cysteine-boundary core sequences can be truncated by about 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 about 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 ActRIIA extracellular domain truncations include SEQ ID NOs: 367 and 368.

[0286] Thus, a general formula for an active portion (e.g., ligand binding) of ActRIIA is a polypeptide comprising, consisting essentially of, or consisting of amino acids 30-110 of SEQ ID NO: 366. Thus, an ActRIIA polypeptide can, for example, begin at a residue corresponding to any one of amino acids 21-30 of SEQ ID NO: 366 (e.g., begin at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and end at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 366 (e.g., begin at 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, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135). Other examples include sequences starting at a position selected from 21 to 30 of SEQ ID NO: 366 (e.g., starting at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), 22 to 30 (e.g., starting at any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), 23 to 30 (e.g., starting at any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30), and 24 to 30 (e.g., starting at any one of amino acids 24, 25, 26, 27, 28, 29, or 30). That is, 111 to 135 of SEQ ID NO: 366 (e.g., ending with any one of 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 to 135 (e.g., ending with any one of amino acids 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),134 or 135), 113 to 135 (e.g., ending with any one of 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 to 135 (e.g., ending with any one of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), 130 to 135 (e.g., ending with any one of amino acids 130, 131, 132, 133, 134, or 135), 111 to 134 (e.g., 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, or 134), 111 to 133 (e.g., 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, or 133), 111 to 132 (e.g., ending with any one of amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and constructs ending at a position selected from the group consisting of 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, and 132) or 111 to 131 (e.g., 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, and 131). Variants within these ranges are also contemplated, particularly those that comprise, consist essentially of, or consist of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding portion of SEQ ID NO: 366. Thus, in some embodiments,ActRIIA polypeptides can comprise, consist essentially of, or consist of 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: 366. ActRIIA polypeptides can also include polypeptides that are 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: 366 and that contain no more than 1, 2, 5, 10, or 15 conservative amino acid changes in the ligand-binding pocket. In some embodiments, an ActRIIA polypeptide of the disclosure begins at a residue corresponding to amino acids 21-30 of SEQ ID NO: 366 (e.g., beginning 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: 366 (e.g., 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, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 18 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135). In some embodiments, an ActRIIA polypeptide comprises, consists of, or consists essentially 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: 366. In certain embodiments, an ActRIIA polypeptide comprises, consists of, or consists essentially 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: 366.In some embodiments, the ActRIIA polypeptide comprises, consists of, or consists essentially 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: 366. In some embodiments, an ActRIIA polypeptide comprises, consists of, or consists essentially 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: 367. In some embodiments, an ActRIIA polypeptide comprises, consists of, or consists essentially 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: 368. In some embodiments, an ActRIIA polypeptide comprises, consists of, or consists essentially 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: 380. In some embodiments, an ActRIIA polypeptide comprises, consists of, or consists essentially 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: 381. In some embodiments, the ActRIIA polypeptide comprises 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: 384, orIn some embodiments, the ActRIIA polypeptide comprises, consists of, or consists essentially 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: 364. In some embodiments, the ActRIIA polypeptide comprises, consists of, or consists essentially 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: 378.

[0287] D) ALK4 polypeptide In certain aspects, the present disclosure relates to ActRII-ALK4 antagonists, including ALK4 polypeptides, including fragments, functional variants, and modified forms thereof, and uses thereof (e.g., treating, preventing, or reducing the rate of progression and / or severity of heart failure (HF) or one or more complications of HF). As used herein, the term "ALK4" refers to any species of the family of activin receptor-like kinase 4 (ALK4) proteins, as well as variant polypeptides derived from such ALK4 proteins by mutagenesis or other modifications (e.g., including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such variant ALK4 polypeptides are provided throughout this disclosure and in International Patent Application Publications WO 2016 / 164089, WO 2016 / 164497, and WO 2018 / 067879, which are incorporated herein by reference in their entireties. Reference to ALK4 herein is understood to refer to any one of the currently identified forms. Members of the ALK4 family are generally transmembrane proteins composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity. Preferably, the ALK4 polypeptide used in accordance with the methods of the present disclosure is soluble. As used herein, the term "soluble ALK4 polypeptide" includes any naturally occurring extracellular domain of an ALK4 polypeptide, as well as any variant thereof (including mutants, fragments, and peptidomimetic forms) that retains useful activity. For example, the extracellular domain of an ALK4 polypeptide binds to a ligand and is generally soluble. An example of a soluble ALK4 polypeptide is the ALK4 extracellular domain shown below (SEQ ID NO: 86). Other examples of soluble ALK4 polypeptides include a signal sequence in addition to the extracellular domain of an ALK4 polypeptide. The signal sequence can be the native signal sequence of the ALK4 polypeptide or a signal sequence from another polypeptide, such as the tissue plasminogen activator (TPA) signal sequence or the honey bee melittin signal sequence.In some embodiments, the ALK4 polypeptide inhibits one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) (e.g., Smad signaling). In some embodiments, the ALK4 polypeptide binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Various exemplary methods and assays for determining the ability of an ALK4 polypeptide to bind to and / or inhibit the activity of one or more ActRII-ALK4 ligands are disclosed herein or otherwise known in the art and can be readily used to determine whether an ActRII-ALK4 polypeptide has the desired binding and / or antagonistic activity. The amino acid numbering of all ALK4-related polypeptides described herein is based on the numbering of the human ALK4 precursor protein sequence (SEQ ID NO: 84) provided below, unless otherwise specified.

[0288] The human ALK4 precursor polypeptide sequence (NCBI RefSeq NP_004293) is as follows:

[0289] [ka]

[0290] Signal peptide Single underscore and the extracellular domain is shown in bold font.

[0291] The processed extracellular human ALK4 polypeptide sequence is as follows:

[0292] [ka]

[0293] The nucleic acid sequence encoding the ALK4 precursor polypeptide is set forth in SEQ ID NO: 221, which corresponds to nucleotides 78 to 1592 of GenBank Reference Sequence NM_004302.4.

[0294] Signal sequence underline and the extracellular domain is in bold.

[0295] [ka] TIFF2024511316000019.tif51141

[0296] The nucleic acid sequence encoding the extracellular ALK4 polypeptide is set forth in SEQ ID NO:222.

[0297] [ka]

[0298] An alternative isoform of the human ALK4 precursor protein sequence, isoform B (NCBI reference sequence NP_064732.3), is as follows:

[0299] [ka]

[0300] The extracellular domain is shown in bold font.

[0301] The processed extracellular ALK4 polypeptide sequence corresponding to isoform B is as follows:

[0302] [ka]

[0303] The nucleic acid sequence encoding the ALK4 precursor protein (isoform B) is shown below (SEQ ID NO: 423), corresponding to nucleotides 186-1547 of GenBank reference sequence NM_020327.3. Nucleotides encoding the extracellular domain are shown in bold font.

[0304] [ka]

[0305] The nucleic acid sequence encoding the extracellular ALK4 polypeptide (isoform B) is as follows:

[0306] [ka]

[0307] An alternative isoform of the human ALK4 precursor polypeptide sequence, isoform C (NCBI Reference Sequence NP_064733.3), is as follows:

[0308] [ka]

[0309] Signal peptide in a single underline and the extracellular domain is shown in bold font.

[0310] The processed extracellular ALK4 polypeptide sequence (isoform C) is as follows:

[0311] [ka]

[0312] The nucleic acid sequence encoding the ALK4 precursor polypeptide (isoform C) is set forth in SEQ ID NO: 223, which corresponds to nucleotides 78 to 1715 of GenBank reference sequence NM_020328.3.

[0313] [ka] TIFF2024511316000028.tif140143

[0314] The nucleic acid sequence encoding the extracellular ALK4 polypeptide (isoform C) is shown in SEQ ID NO:224.

[0315] [ka]

[0316] ALK4 is well conserved among vertebrates, with large stretches of the extracellular domain being completely conserved. For example, Figure 9 shows a multiple sequence alignment of the human ALK4 extracellular domain compared with various ALK4 orthologs. Many of the ligands that bind to ALK4 are also highly conserved. Therefore, from these alignments, it is possible to predict important amino acid positions within the ligand-binding domain that are important for normal ALK4-ligand binding activity, as well as amino acid positions that are likely to tolerate substitutions without significantly altering normal ALK4-ligand binding activity. Thus, active human ALK4 variant polypeptides useful according to the methods of the present disclosure may contain one or more amino acids at corresponding positions from the sequence of another vertebrate ALK4, or may contain residues similar to those in the sequence of a human or other vertebrate.

[0317] Although not meant to be limiting, the following example illustrates this approach to defining active ALK4 variants. As shown in Figure 9, V6 in the human ALK4 extracellular domain (SEQ ID NO: 414) is an isoleucine in Mus muculus ALK4 (SEQ ID NO: 418), so the position may be altered and optionally changed to another hydrophobic residue such as L, I, or F, or a nonpolar residue such as A, as observed in Gallus gallus ALK4 (SEQ ID NO: 417). E40 in the human extracellular domain is K in Gallus gallus ALK4, indicating that this site may tolerate a wide variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, Y, and possibly nonpolar residues such as A. S15 in the human extracellular domain is D in Gallus gallus ALK4, indicating that polar residues such as S, T, R, E, K, H, G, P, G, and Y are preferred, indicating that a wide range of structural variation is tolerated at this position. E40 in the human extracellular domain is K in Gallus gallus ALK4, indicating that charged residues including D, R, K, H, Q, and N are tolerated at this position. R80 in the human extracellular domain is K in star-nosed mole (Condylura cristata) ALK4 (SEQ ID NO: 415), indicating that basic residues including R, K, and H are tolerated at this position. Y77 in the human extracellular domain is F in wild boar (Sus scrofa) ALK4 (SEQ ID NO: 419), indicating that aromatic residues including F, W, and Y are tolerated at this position. P93 in the human extracellular domain is relatively unconserved, appearing as S in Erinaceus europaeus ALK4 (SEQ ID NO: 416) and N in Gallus gallus ALK4; therefore, essentially any amino acid should be tolerated at this position.

[0318] Furthermore, the ALK4 protein has been characterized in the art with respect to its structural and functional characteristics, particularly with respect to ligand binding [e.g., Harrison et al. (2003) J Biol Chem 278(23):21129-21135; Romano et al. (2012) J Mol Model 18(8):3617-3625; and Calvanese et al. (2009) 15(3):175-183]. These references, in addition to the teachings herein, provide ample guidance on how to generate ALK4 variants that retain one or more normal activities (e.g., ligand binding activity).

[0319] For example, a defining structural motif known as the three-finger toxin fold, which is important for ligand binding by type I and type II receptors, is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Thus, the core ligand-binding domain of human ALK4, defined by the outermost of these conserved cysteines, corresponds to positions 34-101 of SEQ ID NO: 84 (ALK4 precursor). The structurally unordered amino acids adjacent to these cysteine-defined core sequences can be truncated by 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, 29, 30, 31, 32, 33 residues at the N-terminus and / or 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 ALK4 extracellular domains for N- and / or C-terminal truncations include SEQ ID NOs: 86, 87, and 422.

[0320] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the ALK4 polypeptides for use disclosed herein (e.g., heteromultimers comprising ALK4 polypeptides and uses thereof) are soluble (e.g., the extracellular domain of ALK4). In other preferred embodiments, the ALK4 polypeptides for use disclosed herein bind to and / or inhibit (antagonize) the activity (e.g., induction of Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, heteromultimers of the disclosure comprise at least one ALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 92, 93, 421, and 422. In some embodiments, heteromultimers of the disclosure consist of or consist essentially of at least one ALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 92, 93, 422.

[0321] In certain aspects, the disclosure relates to heteromultimers comprising an ALK4-Fc fusion polypeptide. In some embodiments, the ALK4-Fc fusion polypeptide begins with any one of amino acids 23-34 (e.g., amino acid residues 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) of SEQ ID NO: 84, 85, or 421, and continues with any one of amino acids 101-126 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 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, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126). In some embodiments, the ALK4-Fc fusion polypeptide comprises an ALK4 domain 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 amino acids 34-101 of SEQ ID NO: 84, 85, or 421. In some embodiments, the ALK4-Fc fusion polypeptide comprises an ALK4 domain 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 amino acids 23-126 of SEQ ID NO: 84, 85, or 421. In some embodiments, the ALK4-Fc fusion polypeptide comprises an ALK4 domain 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 the amino acid sequence of any one of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, 422.

[0322] E) ALK7 polypeptide In certain aspects, the present disclosure relates to ActRII-ALK4 antagonists comprising ALK7 polypeptides, including fragments, functional variants, and modified forms thereof, and uses thereof (e.g., treating, preventing, or reducing the rate of progression and / or severity of heart failure (HF) or one or more complications of HF). As used herein, the term "ALK7" refers to any species of the family of activin receptor-like kinase 7 (ALK7) proteins, as well as variant polypeptides derived from such ALK7 proteins by mutagenesis or other modifications (e.g., including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such variant ALK7 polypeptides are provided throughout this disclosure and in International Patent Application Publications WO 2016 / 164089 and WO 2016 / 164503, which are incorporated herein by reference in their entireties. References herein to ALK7 are understood to refer to any one of the currently identified forms. ALK7 family members are generally all-transmembrane polypeptides, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase specificity. The amino acid sequence of human ALK7 precursor polypeptide is shown below (SEQ ID NO: 120). Preferably, the ALK7 polypeptide used in accordance with the methods of the present disclosure is soluble. As used herein, the term "soluble ALK7 polypeptide" includes any naturally occurring extracellular domain of an ALK7 polypeptide, as well as any variant thereof (including mutants, fragments, and peptidomimetic forms) that retains useful activity. For example, the extracellular domain of an ALK7 polypeptide binds to a ligand and is generally soluble. An example of a soluble ALK7 polypeptide is the following ALK7 extracellular domain (SEQ ID NO: 123): Other examples of soluble ALK7 polypeptides include a signal sequence in addition to the extracellular domain of an ALK7 polypeptide. The signal sequence can be the native signal sequence of ALK7 or a signal sequence from another polypeptide, such as the tissue plasminogen activator (TPA) signal sequence or the honeycomb melittin signal sequence.In some embodiments, the ALK7 polypeptide inhibits one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) (e.g., Smad signaling). In some embodiments, the ALK7 polypeptide binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Various exemplary methods and assays for determining the ability of an ALK7 polypeptide to bind to and / or inhibit the activity of one or more ActRII-ALK4 ligands are disclosed herein or otherwise known in the art and can be readily used to determine whether an ALK7 polypeptide has the desired binding and / or antagonistic activity. The amino acid numbering of all ALK7-related polypeptides described herein is based on the numbering of the human ALK7 precursor protein sequence (SEQ ID NO: 120) provided below, unless otherwise specified.

[0323] Four naturally occurring isoforms of human ALK7 have been described. The sequence of the human ALK7 isoform 1 precursor polypeptide (NCBI Reference Sequence NP_660302.2) is as follows:

[0324] [ka]

[0325] Signal peptide Single underscore and the extracellular domain is shown in bold font.

[0326] The processed extracellular ALK7 isoform 1 polypeptide sequence is as follows:

[0327] [ka]

[0328] The nucleic acid sequence encoding the human ALK7 isoform 1 precursor polypeptide is shown below in SEQ ID NO: 233, which corresponds to nucleotides 244 to 1722 of GenBank reference sequence NM_145259.2.

[0329] [ka] TIFF2024511316000033.tif58141

[0330] The nucleic acid sequence encoding the processed extracellular ALK7 polypeptide (isoform 1) is shown in SEQ ID NO:234.

[0331] [ka]

[0332] The amino acid sequence of an alternative isoform of human ALK7, isoform 2 (NCBI Reference Sequence NP_001104501.1), in its processed form is shown below (SEQ ID NO: 124), with the extracellular domain shown in bold.

[0333] [ka]

[0334] The amino acid sequence of the extracellular ALK7 polypeptide (isoform 2) is as follows:

[0335] [ka]

[0336] The nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 2) is shown below in SEQ ID NO: 235, which corresponds to nucleotides 279 to 1607 of the NCBI reference sequence NM_001111031.1.

[0337] [ka]

[0338] The nucleic acid sequence encoding the extracellular ALK7 polypeptide (isoform 2) is set forth in SEQ ID NO:236.

[0339] [ka]

[0340] The amino acid sequence of another human ALK7 precursor polypeptide, isoform 3 (NCBI Reference Sequence NP_001104502.1), is shown below (SEQ ID NO: 121). The signal peptide is Single underscore is shown.

[0341] [ka]

[0342] The amino acid sequence of the processed ALK7 polypeptide (isoform 3) is as follows (SEQ ID NO: 126). This isoform lacks a transmembrane domain and is therefore proposed to be entirely soluble (Roberts et al., 2003, Biol Reprod 68:1719-1726). N-terminal variants of SEQ ID NO: 126 are predicted as described below.

[0343] [ka]

[0344] The nucleic acid sequence encoding the unprocessed ALK7 polypeptide precursor polypeptide (isoform 3) is shown in SEQ ID NO: 237 and corresponds to nucleotides 244 to 1482 of the NCBI reference sequence NM_001111032.1.

[0345] [ka]

[0346] The nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 3) is shown in SEQ ID NO:238.

[0347] [ka]

[0348] The amino acid sequence of another human ALK7 precursor polypeptide, isoform 4 (NCBI Reference Sequence NP_001104503.1), is shown below (SEQ ID NO: 122). The signal peptide is Single underscore is shown.

[0349] [ka]

[0350] The amino acid sequence of the processed ALK7 polypeptide (isoform 4) is as follows (SEQ ID NO: 127). Like ALK7 isoform 3, isoform 4 lacks the transmembrane domain and is therefore proposed to be soluble in its entirety (Roberts et al., 2003, Biol Reprod 68:1719-1726). N-terminal variants of SEQ ID NO: 127 are predicted as described below.

[0351] [ka]

[0352] The nucleic acid sequence encoding the unprocessed ALK7 polypeptide precursor polypeptide (isoform 4) is set forth in SEQ ID NO: 239 and corresponds to nucleotides 244 to 1244 of the NCBI reference sequence NM_001111033.1.

[0353] [ka] TIFF2024511316000046.tif65141

[0354] The nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 4) is shown in SEQ ID NO:240.

[0355] [ka]

[0356] Based on the signal sequence of rat full-length ALK7 (isoform 1) (see NCBI reference sequence NP_620790.1) and the high degree of sequence identity between human and rat ALK7, the processed form of human ALK7 isoform 1 is predicted to be as follows (sequence number 128):

[0357] [ka]

[0358] SEQ ID NO: 123 predicts active variants of processed ALK7 isoform 1 truncated at the N-terminus by 1, 2, 3, 4, 5, 6, or 7 amino acids, and SEQ ID NO: 128 predicts active variants of processed ALK7 isoform 1 truncated at the N-terminus by 1 or 2 amino acids. Consistent with SEQ ID NO: 128, leucine is further predicted to be the N-terminal amino acid of the processed forms of human ALK7 isoform 3 (SEQ ID NO: 126) and human ALK7 isoform 4 (SEQ ID NO: 127).

[0359] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK7 polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the ALK7 polypeptides for use in accordance with the present disclosure (e.g., heteromultimers comprising ALK7 polypeptides and uses thereof) are soluble (e.g., the extracellular domain of ALK7). In other preferred embodiments, the ALK7 polypeptides for use in accordance with the present disclosure bind to one or more ActRII-ALK4 ligands. Thus, in some preferred embodiments, the ALK7 polypeptides for use in accordance with the present disclosure inhibit (antagonize) the activity of one or more ActRII-ALK4 ligands (e.g., induction of Smad signaling). In some embodiments, heteromultimers of the present disclosure comprise at least one ALK7 polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 255, 133, and 134. In some embodiments, heteromultimers of the present disclosure consist of or consist essentially of at least one ALK7 polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 255, 133, and 134.

[0360] ALK7 is well conserved among vertebrates, with large stretches of the extracellular domain being completely conserved. For example, Figure 22 shows a multiple sequence alignment of the human ALK7 extracellular domain compared to various ALK7 orthologs. From these alignments, it is therefore possible to predict key amino acid positions within the ligand-binding domain that are important for normal ALK7-ligand binding activity, as well as amino acid positions that are likely to tolerate substitutions without significantly altering normal ALK7-ligand binding activity. Thus, active human ALK7 variant polypeptides useful according to the methods of the present disclosure may contain one or more amino acids at corresponding positions from the sequence of another vertebrate ALK7, or may contain residues similar to those in human or other vertebrate sequences. While not meant to be limiting, the following examples illustrate this approach to defining active ALK7 variants. Since V61 in the human ALK7 extracellular domain (SEQ ID NO: 425) is an isoleucine in common marmoset (Callithrix jacchus) ALK7 (SEQ ID NO: 428), the position may be altered, possibly to another hydrophobic residue such as L, I, or F, or to a non-polar residue such as A. L32 in the human extracellular domain is R in Philippine tarsier (Tarsius syrichta) (SEQ ID NO: 429) ALK7, 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, Y, and non-polar groups such as A; K37 in the human extracellular domain is R in chimpanzee (Pan troglodytes) ALK7 (SEQ ID NO: 426), indicating that basic residues including R, K, and H are tolerated at this position; and P4 in the human extracellular domain is relatively less conserved and appears as A in chimpanzee ALK7, indicating that a wide variety of amino acids should be tolerated at this position.

[0361] Furthermore, the ALK7 protein has been characterized in the art with respect to its structural and functional features [e.g., Romano et al. (2012) Journal of Molecular Modeling 18(8):3617-3625]. For example, a defining structural motif known as the three-finger toxin fold, which is important for ligand binding by type I and type II receptors, is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor [Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870]. Thus, the core ligand-binding domain of human ALK7, defined by the outermost of these conserved cysteines, corresponds to positions 28-92 of SEQ ID NO: 120. The structurally unordered amino acids adjacent to these cysteine-defined core sequences can be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 residues at the N-terminus and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 residues at the C-terminus without necessarily altering ligand binding. Exemplary ALK7 extracellular domains for N- and / or C-terminal truncations include SEQ ID NOs: 123, 125, 126, and 127.

[0362] Thus, a general formula for an active portion (e.g., a ligand-binding portion) of ALK7 includes amino acids 28-92 of SEQ ID NO: 120. Thus, an ALK7 polypeptide can, for example, begin at a residue corresponding to any one of amino acids 20-28 of SEQ ID NO: 120 (e.g., begin at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, or 28) and end at a position corresponding to any one of amino acids 92-113 of SEQ ID NO: 120 (e.g., begin at amino acids 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, or 128). 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113).

[0363] Other examples include sequences starting at positions 21 to 28 (e.g., any of positions 21, 22, 23, 24, 25, 26, 27, or 28), 24 to 28 (e.g., any of positions 24, 25, 26, 27, or 28), or 25 to 28 (e.g., any of positions 25, 26, 27, or 28) of SEQ ID NO: 120 and continuing through positions 93 to 112 (e.g., positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, or 228) of SEQ ID NO: 120. 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110), 93-100 (e.g., any of positions 93, 94, 95, 96, 97, 98, 99, or 100), or 93-95 (e.g., any of positions 93, 94, or 95). Variants within these ranges are also contemplated, particularly variants having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ ID NO: 120.

[0364] The variations described herein can be combined in various ways. In some embodiments, the ALK7 variant contains no more than 1, 2, 5, 6, 7, 8, 9, 10, or 15 conservative amino acid changes in the ligand-binding pocket. Sites outside the binding pocket where variability may be particularly well tolerated include the amino and carboxy termini of the extracellular domain (as described above).

[0365] F) Follistatin polypeptide In other embodiments, the ActRII-ALK4 antagonist is a follistatin polypeptide. As described herein, follistatin polypeptides can be used to treat, prevent, or reduce the rate of progression and / or severity of heart failure associated with metabolic complications (e.g., diabetes, obesity), particularly to treat, prevent, or reduce the rate of progression and / or severity of one or more heart failure-related complications.

[0366] The term "follistatin polypeptide" includes polypeptides comprising any naturally occurring polypeptide of follistatin and any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity, and further includes any functional monomer or multimer of follistatin. In certain preferred embodiments, the follistatin polypeptides of the present disclosure bind to and / or inhibit activin activity, particularly activin A. Follistatin polypeptide variants that retain activin-binding properties can be identified based on previous studies involving the interaction of follistatin with activin. For example, WO 2008 / 030367 discloses specific follistatin domains ("FSDs") shown to be important for activin binding. As shown in SEQ ID NOS: 392-394 below, the follistatin N-terminal domain ("FSND" SEQ ID NOS: 392), FSD2 (SEQ ID NOS: 394), and, to a lesser extent, FSD1 (SEQ ID NOS: 393) represent exemplary domains within follistatin that are important for activin binding. Additionally, methods for generating and testing libraries of polypeptides are described above in the context of ActRII polypeptides, and such methods also relate to generating and testing variants of follistatin. Follistatin polypeptides include polypeptides having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and optionally derived from any known follistatin sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Exemplary follistatin polypeptides include mature follistatin polypeptides or shorter isoforms or other variants of the human follistatin precursor polypeptide (SEQ ID NO: 390), e.g., as described in WO 2005 / 025601.

[0367] The human follistatin precursor polypeptide isoform FST344 is as follows:

[0368] [ka]

[0369] Signal peptide underline Also, on top underline Underlined are the last 27 residues representing the C-terminal extension that distinguishes this follistatin isoform from the shorter follistatin isoform FST317 shown below.

[0370] The human follistatin precursor polypeptide isoform FST317 is as follows:

[0371] [ka]

[0372] Signal peptide underline is attached.

[0373] The follistatin N-terminal domain (FSND) sequence is as follows:

[0374] [ka]

[0375] The FSD1 and FSD2 sequences are as follows:

[0376] [ka] [ka]

[0377] G) Fusion Polypeptides In certain aspects, the present disclosure provides ActRII-ALK4 antagonists that are fusion polypeptides. Fusion polypeptides can be prepared according to any of the methods disclosed herein or known in the art.

[0378] In some embodiments, any of the fusion polypeptides disclosed herein comprises the following components: a) any of the polypeptides disclosed herein ("A") (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), b) any of the linkers disclosed herein ("B"), c) any of the heterologous moieties disclosed herein ("C") (e.g., an Fc immunoglobulin domain), and optionally a leader sequence ("X") (e.g., a tissue plasminogen activator leader sequence). In such embodiments, the fusion polypeptide can be arranged (N-terminus to C-terminus) as follows: ABC or CBA. In such embodiments, the fusion polypeptide can be arranged (N-terminus to C-terminus) as follows: XABC or XCBA. In some embodiments, the fusion polypeptide comprises each of A, B, and C (and optionally a leader sequence) and up to 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 additional amino acids (but may include further post-translational modifications such as glycosylation).

[0379] In some embodiments, the fusion polypeptide comprises a leader sequence arranged (N-terminus to C-terminus) as follows: XABC, wherein the fusion polypeptide comprises 1, 2, 3, 4, or 5 amino acids between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence arranged (N-terminus to C-terminus) as follows: XCBA, wherein the fusion polypeptide comprises 1, 2, 3, 4, or 5 amino acids between X and C. In some embodiments, the fusion polypeptide comprises a leader sequence arranged (N-terminus to C-terminus) as follows: XABC, wherein the fusion polypeptide comprises an alanine between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence arranged (N-terminus to C-terminus) as follows: XCBA, wherein the fusion polypeptide comprises an alanine between X and C. In so...

Claims

1. A pharmaceutical composition for treating heart failure associated with diabetic cardiomyopathy in a patient in need thereof, comprising an ActRIIB-Fc-ALK4-Fc antagonist.

2. The method of claim 1 , wherein the patient is obese.

3. The pharmaceutical composition of claim 1 or 2, wherein the patient has diabetes.

4. The pharmaceutical composition according to claim 1 or 2, wherein the heart failure is heart failure associated with preserved ejection fraction (HFpEF).

5. The pharmaceutical composition of claim 1 or 2, wherein the patient has left ventricular (LV) hypertrophy.

6. The pharmaceutical composition of claim 1 or 2, which reduces LV hypertrophy in said patient.

7. The pharmaceutical composition of claim 1 or 2, wherein the patient has diastolic dysfunction.

8. The pharmaceutical composition of claim 1 or 2, which improves diastolic dysfunction in the patient.

9. 3. The pharmaceutical composition of claim 1 or 2, wherein the patient has decreased ventricular relaxation and increased filling pressures.

10. 3. The pharmaceutical composition of claim 1 or 2, which increases ventricular relaxation and decreases filling pressures in said patient.

11. 3. The pharmaceutical composition of claim 1 or 2, which reduces a patient's ratio of early diastolic transmitral flow to early diastolic mitral annular tissue velocity (E / e' ratio).

12. 3. The pharmaceutical composition of claim 1 or 2, which reduces brain natriuretic peptide (BNP) levels in the patient.

13. 3. The pharmaceutical composition of claim 1 or 2, wherein the ActRIIB-Fc-ALK4-Fc antagonist is a polypeptide heteromultimer comprising: (i) an ActRIIB-Fc fusion polypeptide comprising an ActRIIB polypeptide domain and an Fc domain; and (ii) an ALK4-Fc fusion polypeptide comprising an ALK4 polypeptide domain and an Fc domain.

14. The pharmaceutical composition of claim 13, wherein the ALK4 polypeptide domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421 and 422.

15. The pharmaceutical composition of claim 13, wherein the ALK4-Fc fusion polypeptide further comprises a linker domain located between the ALK4 polypeptide domain and the Fc domain.

16. 16. The pharmaceutical composition of claim 15, wherein the linker domain is selected from TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.

17. 14. The pharmaceutical composition of claim 13, wherein the ActRIIB polypeptide domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence beginning with any one of amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of SEQ ID NO:2 and ending with any one of amino acid residues 109, 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 134 of SEQ ID NO:

2.

18. 14. The pharmaceutical composition of claim 13, wherein the ActRIIB polypeptide domain comprises an amino acid sequence that is at least 90% identical to amino acids 29-109, 25-131, or 20-134 of SEQ ID NO:2, or at least 90% identical to the amino acid sequence of SEQ ID NO:53, 388, or 389.

19. The pharmaceutical composition of claim 13, wherein the ActRIIB-Fc fusion polypeptide further comprises a linker domain disposed between the ActRIIB polypeptide domain and the Fc domain.

20. 20. The pharmaceutical composition of claim 19, wherein the linker domain is selected from TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.

21. 20. The pharmaceutical composition of claim 19, wherein the ActRIIB-Fc fusion polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

12.

22. The ActRIIB polypeptide domain comprises one or more of the following amino acid sequences with respect to the amino acid sequence of SEQ ID NO:2: A24N, S26T, N35E, E37A, E37D, L38N, R40A, R40K, S44T, L46V, L46I, L46F, L46A, E50K, E50P, E50L, E52A, E52D, E52G, E52H, E52K, E52N, E52P, E52R, E52S, E52T, E52Y, Q53R, Q53K, Q53N, Q53H, D54T ... A, K55A, K55D, K55E, K55R, R56A, L57E, L57I, L57R, L57T, L57V, Y60D, Y60F, Y60K, Y60P, R64A, R64H, R64K, R64N, N65A, S67N, S67T, G68R, K74A, K74E, K74F, K74I, K74R, K74Y, W78A, W78Y, L79A, L79D, L79E, L79F, L79H, L79K, L79P, L79R, L79 S, L79T, L79W, D80A, D80F, D80G, D80I, D80K, D80M, D80N, D80R, F82A, F82D, F82E, F82I, F82K, F82L, F82S, F82T, F82W, F82Y, N83A, N83R, T93D, T93E, T93G, T93H, T93K, T93P, T93R, T93S, T93Y, E94K, Q98D, Q98E, Q98K, Q98R, V99E, V99G, V99 14. The pharmaceutical composition of claim 13, comprising one or more amino acid substitutions selected from the group consisting of K, E105N, F108I, F108L, F108V, F108Y, E111D, E111H, E111K, 111N, E111Q, E111R, R112H, R112K, R112N, R112S, R112T, A119P, A119V, G120N, E123N, P129N, P129S, P130A, P130R, and A132N.

23. 22. The pharmaceutical composition of claim 21 , wherein the ActRIIB polypeptide domain comprises one or more amino acid substitutions selected from the group consisting of L38N, E50L, E52D, E52N, E52Y, L57E, L57I, L57R, L57T, L57V, Y60D, G68R, K74E, W78Y, L79E, L79F, L79H, L79R, L79S, L79T, L79W, F82D, F82E, F82I, F82K, F82L, F82S, F82T, F82Y, N83R, E94K, and V99G with respect to the amino acid sequence of SEQ ID NO:2.

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