ACTRII-ALK4 ANTAGONISTS AND METHODS OF TREATING HEART FAILURE

Abstract

In some aspects, the 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 treating, preventing or reducing the progression rate and/or severity of one or more HF-associated comorbidities. The disclosure also provides methods of using an ActRII-ALK4 antagonist to treat, prevent, or reduce the progression rate and/or severity of heart failure associated with a variety of conditions including, but not limited to, genetic cardiomyopathies, muscle wasting diseases, and muscular dystrophies.

Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 15, 2022, is named 1848179-150-301_Seq.txt and is 425,145 bytes in size.

BACKGROUND

The prevalence of heart failure (HF) depends on the definition applied, but it affects approximately 1-2% of the adult population in developed countries, rising to ≥10% among people 70 years of age. Among people 65 years of age presenting to primary care with breathlessness on exertion, one in six will have unrecognized HF (mainly HFpEF). The lifetime risk of HF at age 55 years is 33% for men and 28% for women. The proportion of patients with HFpEF ranges from 22 to 73%, depending on the definition applied, the clinical setting (primary care, hospital clinic, and hospital admission), age and sex of the studied population, previous myocardial infarction and the year of publication.

Dilated cardiomyopathy, one of many genetic cardiomyopathies involved in heart failure, is defined by the presence of left ventricular dilatation and contractile dysfunction. Genetic mutations involving genes that encode cytoskeletal, sarcomere, and nuclear envelope proteins, among others, account for up to 35% of cases. The most common presenting symptoms relate to congestive heart failure, but can also include circulatory collapse, arrhythmias, and thromboembolic events. The prognosis is worst for individuals with the lowest ejection fractions or severe diastolic dysfunction. Treatment of chronic heart failure comprises general heart failure medications that improve survival and reduce hospital admission, namely, angiotensin converting enzyme inhibitors and 3 blockers.

Therefore, there is a high, unmet need for effective therapies for treating heart failure (e.g., genetic cardiomyopathies, including DCM). Accordingly, it is an object of the present disclosure to provide methods for treating, preventing, or reducing the progression rate and/or severity of heart failure, particularly treating, preventing or reducing the progression rate and/or severity of one or more heart failure-associated comorbidities.

SUMMARY

As demonstrated herein, an ActRII-ALK4 antagonist is effective in treating heart failure. In particular, an ActRIIB-ALK4 heterodimer protein demonstrated cardio-protective effects in a murine Mdx model of heart failure associated with reduced ejection fraction. For example, data presented herein shows that treatment with an ActRIIB-ALK4 heterodimer has positive effects on various complications associated with this heart failure model including, but not limited to, LV contractility, hypertrophy, LV wall thickness, heart weight, systolic function, and serum biomarkers of cardiac injury (e.g., cTnI serum levels). While not wishing to be bound to any particular mechanism, it is expected that the effects of the ActRIIB-ALK4 heterodimer on heart failure is caused primarily by antagonizing ligand-signaling as 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 ligands” or “ActRII-ALK4 ligand”). Regardless of the mechanism, it is apparent from the data presented herein that ActRIIB-ALK4 heterodimers have significant positive effects in ameliorating various complications associated with heart failure and further suggests that other ActRII-ALK4 antagonists may also be useful in treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies).

As disclosed herein, the term “ActRII-ALK4 antagonist” refers a variety of agents that may 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 Smads 2 and 3). ActRII-ALK4 antagonists to be used in accordance with the methods and uses of the disclosure include a variety of forms, for example, ActRII-ALK4 ligand traps (e.g., soluble ActRIIA polypeptides or ActRIIB polypeptides including variants as well as heteromultimers and homomultimers thereof), ActRII-ALK4 antibody antagonists (e.g., 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).

In certain aspects, the disclosure provides ActRII-ALK4 antagonists comprising soluble ActRIIB, ActRIIA, ALK4, ALK7, or follistatin polypeptides to antagonize the signaling of ActRII-ALK4 ligands generally, in any process associated with heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). ActRII-ALK4 antagonists of the disclosure may antagonize one or more ligands of ActRII-ALK4, such as activin A, activin B, GDF8, GDF11, BMP6, or BMP10, and may therefore be useful in treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more comorbidities of heart failure (e.g. arterial hypertension, atrial fibrillation, cognitive dysfunction, diabetes, hypercholesterolemia, iron deficiency, kidney dysfunction, metabolic syndrome, obesity, physical deconditioning, potassium disorders, pulmonary disease (e.g., COPD), and sleep apnea).

In certain aspects, an ActRII-ALK4 antagonist to be 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 (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure) is an ActRII-ALK4 ligand trap polypeptide antagonist including variants thereof as well as 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, that are capable of binding to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Therefore, an ActRII-ALK4 ligand trap generally includes polypeptides that are 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 family of type II activin receptors. 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, as well has homomultimers (e.g., ActRIIB homodimers) and heteromultimers (e.g., ActRIIB-ALK4 or ActRIIB-ALK7 heterodimers). In some embodiments, an ActRII-ALK4 ligand trap comprises an ActRIIA polypeptide, including variants thereof, as well has homomultimers (e.g., ActRIIA homodimers) and heteromultimers (e.g., ActRIIA-ALK4 or ActRIIA-ALK7 heterodimers). In other embodiments, an ActRII-ALK ligand trap comprises a soluble ligand trap protein including, but not limited to, or a follistatin polypeptide as well as variants thereof. In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 antibody antagonist. In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 small molecule antagonist. In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 polynucleotide antagonist.

In part, the disclosure provides methods of treating heart failure associated with dilated cardiomyopathy (DCM), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. The disclosure also provides methods of treating, preventing, or reducing the progression rate and/or severity of one or more comorbidities of heart failure associated with dilated cardiomyopathy (DCM), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist.

In some embodiments, dilated cardiomyopathy is a genetic form of DCM. In some embodiments, dilated cardiomyopathy is selected from the group consisting of autosomal recessive DCM, X-linked DCM, and mitochondrial DCM.

In some embodiments, the dilated cardiomyopathy is associated with Duchenne Muscular Dystrophy (DMD). In some embodiments, dilated cardiomyopathy is associated with one or more mutations in the dystrophin (DMD) gene.

In some embodiments of the present disclosure, a patient has HFrEF heart failure. In some embodiments, a patient is also administered one or more agents selected from the group consisting of stop codon read-through therapies, viral vector-based gene therapies, antisense oligonucleotides (AON) therapies for exon skipping, Atalurenhas, utrophin overexpression therapies, tadalafil, myostatin inhibitors, and cell therapies. In some embodiments, a patient is also administered one or more agents selected from the group consisting of rAAV2.5-CMV-minidystrophin, SGT-001, rAAVrh74.MHCK7.micro-Dystrophin, SRP-9001, and GALGT2. In some embodiments, a patient is also administered one or more agents selected from the group consisting of eteplirsen (SRP-4051), golodirsen (SRP-4053), casimersen (SRP-4045), peptide-conjugated eteplirsen (SRP-5051), SRP-5053, SRP-5045, SRP-5052, SRP-5044, SRP-5050, viltolarsen (NS-065/NCNP-01), NS-089/NCNP-02 (exon skipping 44), DS-5141b (exon skipping 45), suvodirsen (WVE-210,201), drisapersen (PRO051), PNA-ssODN, M12-PMO (exon 23 skipping), and M12-PMO (exon 10 skipping). In some embodiments, a patient is also administered eteplirsen. In some embodiments, a patient is also administered golodirsen. In some embodiments, a patient is also administered casimersen. In some embodiments, a patient is also administered viltolarsen. In some embodiments, a patient is also administered peptide-conjugated eteplirsen. In some embodiments, a patient is also administered suvodirsen. In some embodiments, a patient is also administered drisapersen.

In part, the disclosure provides methods of treating heart failure associated with a muscle wasting disease, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. The disclosure also provides methods of treating, preventing, or reducing the progression rate and/or severity of one or more comorbidities of heart failure associated with a muscle wasting disease, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. In some embodiments of the present disclosure, a patient has HFrEF heart failure.

In some embodiments of the present disclosure, the muscle wasting disease is a muscular dystrophy. In some embodiments, the muscle wasting disease is a muscular dystrophy selected from the group consisting of Becker muscular dystrophy (BMD), Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), and Friedreich's ataxia muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne Muscular Dystrophy (DMD). In some embodiments, the muscular dystrophy is associated with one or more mutations in the dystrophin (DMD) gene.

In some embodiments of the present disclosure, a patient is also administered one or more agents selected from the group consisting of stop codon read-through therapies, viral vector-based gene therapies, antisense oligonucleotides (AON) therapies for exon skipping, Atalurenhas, utrophin overexpression therapies, tadalafil, myostatin inhibitors, and cell therapies. In some embodiments, a patient is also administered one or more agents selected from the group consisting of rAAV2.5-CMV-minidystrophin, SGT-001, rAAVrh74.MHCK7.micro-Dystrophin, SRP-9001, and GALGT2. In some embodiments, a patient is also administered one or more of agents selected from the group consisting of eteplirsen (SRP-4051), golodirsen (SRP-4053), casimersen (SRP-4045), peptide-conjugated eteplirsen (SRP-5051), SRP-5053, SRP-5045, SRP-5052, SRP-5044, SRP-5050, viltolarsen (NS-065/NCNP-01), NS-089/NCNP-02 (exon skipping 44), DS-5141b (exon skipping 45), suvodirsen (WVE-210,201), drisapersen (PR0051), PNA-ssODN, M12-PMO (exon 23 skipping), and M12-PMO (exon 10 skipping). In some embodiments, a patient is also administered eteplirsen. In some embodiments, a patient is also administered golodirsen. In some embodiments, a patient is also administered casimersen. In some embodiments, a patient is also administered viltolarsen. In some embodiments, a patient is also administered peptide-conjugated eteplirsen. In some embodiments, a patient is also administered suvodirsen. In some embodiments, a patient is also administered drisapersen.

In some embodiments of the present disclosure, the muscle wasting disease is associated with one of more of disorders selected from the group consisting of muscle atrophies (e.g., Post-Polio Muscle Atrophy (PPMA)), cachexias (e.g., cardiac cachexia, AIDS cachexia, and cancer cachexia), malnutrition, leprosy, diabetes, renal disease, Chronic Obstructive Pulmonary Disease (COPD), cancer, end stage renal failure, sarcopenia, emphysema, osteomalacia, HIV infection, and AIDS.

In some embodiments of the present disclosure, the muscular dystrophy is Limb Girdle Muscular Dystrophy (LGMD). In some embodiments of the present disclosure, the muscular dystrophy is associated with one or more mutations in a gene selected from the group consisting of myotilin (MYOT), lamin A/C (LMNA), Caveolin-3 (CA V3), Calpain-3 (CAPN3), Dysferlin (DYSF), γ-Sarcoglycan (SGCG), α-Sarcoglycan (SGCA), β-Sarcoglycan (SGCB), and/or δ-Sarcoglycan (SGCD), fukutin-related protein (FKRP), Anoctamin-5 (ANO5). In some embodiments of the present disclosure, a patient is also administered one or more of agents selected from the group consisting of SRP-9003, SRP-9004, SRP-9005, SRP-6004, SRP-9006, and LGMD2A.

In some embodiments of the present disclosure, the muscular dystrophy is Friedreich's ataxia muscular dystrophy. In some embodiments, the muscular dystrophy is associated with one or more mutations in the frataxia gene (FXN).

In some embodiments of the present disclosure, the muscular dystrophy is Myotonic dystrophy. In some embodiments, the muscular dystrophy is associated with one or more mutations in a gene selected from the group consisting of myotonic dystrophy protein kinase (DMPK) and CCHC-type zinc finger nucleic acid binding protein (CNBP) gene.

In part, the disclosure provides methods of treating heart failure associated with genetic cardiomyopathies, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. The disclosure also provides methods of treating, preventing, or reducing the progression rate and/or severity of one or more comorbidities of heart failure associated with genetic cardiomyopathies, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist.

In some embodiments of the present disclosure, the genetic cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic cardiomyopathy, left ventricular noncompaction cardiomyopathy, and restrictive cardiomyopathy. In some embodiments, the genetic cardiomyopathy is dilated cardiomyopathy.

In part, the disclosure provides methods of treating heart failure (HF), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist. The disclosure also provides methods of treating, preventing, or reducing the progression rate and/or severity of one or more comorbidities of heart failure, comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist.

In some embodiments, the heart failure is a genetic cardiomyopathy. In some embodiments, the heart failure is a dilated cardiomyopathy (DCM). In some embodiments, the heart failure is associated with Duchenne muscular dystrophy (DMD). In some embodiments, the heart failure is associated with one or more mutations in the dystrophin (DMD) gene.

In some embodiments of the disclosure, a patient is also administered one or more agents selected from the group consisting of stop codon read-through therapies, viral vector-based gene therapies, antisense oligonucleotides (AON) therapies for exon skipping, Atalurenhas, utrophin overexpression therapies, tadalafil, myostatin inhibitors, and cell therapies. In some embodiments, a patient is also administered one or more agents selected from the group consisting of rAAV2.5-CMV-minidystrophin, SGT-001, rAAVrh74.MHCK7.micro-Dystrophin, SRP-9001, and GALGT2. In some embodiments, a patient is also administered one or more agents selected from the group consisting of eteplirsen (SRP-4051), golodirsen (SRP-4053), casimersen (SRP-4045), peptide-conjugated eteplirsen (SRP-5051), SRP-5053, SRP-5045, SRP-5052, SRP-5044, SRP-5050, viltolarsen (NS-065/NCNP-01), NS-089/NCNP-02 (exon skipping 44), DS-5141b (exon skipping 45), suvodirsen (WVE-210,201), drisapersen (PRO051), PNA-ssODN, M12-PMO (exon 23 skipping), and M12-PMO (exon 10 skipping). In some embodiments, a patient is also administered eteplirsen. In some embodiments, a patient is also administered golodirsen. In some embodiments, a patient is also administered casimersen. In some embodiments, a patient is also administered viltolarsen. In some embodiments, a patient is also administered peptide-conjugated eteplirsen. In some embodiments, a patient is also administered suvodirsen. In some embodiments, a patient is also administered drisapersen.

In some embodiments of the present disclosure, the heart failure is associated with Limb Girdle Muscular Dystrophy (LGMD). In some embodiments, the heart failure is associated with one or more mutations in a gene selected from the group consisting of myotilin (MYOT), lamin A/C (LMNA), Caveolin-3 (CA V3), Calpain-3 (CAPN3), Dysferlin (DYSF), γ-Sarcoglycan (SGCG), α-Sarcoglycan (SGCA), β-Sarcoglycan (SGCB), and/or δ-Sarcoglycan (SGCD), fukutin-related protein (FKRP), Anoctamin-5 (ANO5). In some embodiments, a patient is also administered one or more agents selected from the group consisting of SRP-9003, SRP-9004, SRP-9005, SRP-6004, SRP-9006, and LGMD2A.

In some embodiments of the present disclosure, the heart failure is associated with Friedreich's ataxia muscular dystrophy. In some embodiments, the heart failure is associated with one or more mutations in the frataxin gene (FXN).

In some embodiments of the present disclosure, the heart failure is associated with Myotonic dystrophy. In some embodiments, the heart failure is associated with one or more mutations in a gene selected from the group consisting of myotonic dystrophy protein kinase (DMPK) and CCHC-type zinc finger nucleic acid binding protein (CNBP) gene.

In some embodiments of the present disclosure, the heart failure is associated with Hypertrophic cardiomyopathy (HCM). In some embodiments, the heart failure is associated with Arrhythmogenic cardiomyopathy (AC). In some embodiments, the heart failure is associated with Left ventricular noncompaction cardiomyopathy (LVNC). In some embodiments, the heart failure is associated with Restrictive cardiomyopathy (RC).

In some embodiments of the present disclosure, the heart failure is heart failure with preserved ejection fraction (HFpEF). In some embodiments, a patient has normal LVEF and an LVEF of ≥50%. In some embodiments, a patient has elevated levels of natriuretic peptides.

In some embodiments of the present disclosure, the heart failure is heart failure with reduced ejection fraction (HFrEF). In some embodiments, a patient has reduced LVEF and an LVEF of <40%.

In some embodiments of the present disclosure, the heart failure is heart failure with heart failure with mid-range ejection fraction (HFmrEF). In some embodiments, a patient has mid-range LVEF and an LVEF of between about 40% and about 49%. In some embodiments, a patient has elevated levels of natriuretic peptides.

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

In some embodiments, methods of the present disclosure reduce a patient's NYHA Class. In some embodiments, the method reduces a patient's NYHA Class from Class IV to Class III. In some embodiments, the method reduces a patient's NYHA Class from Class IV to Class II. In some embodiments, the method reduces a patient's NYHA Class from Class IV to Class I. In some embodiments, the method reduces a patient's NYHA Class from Class III to Class II. In some embodiments, the method reduces a patient's NYHA Class from Class III to Class I. In some embodiments, the method reduces a patient's NYHA Class from Class II to Class I.

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

In some embodiments, methods of the present disclosure reduce a patient's ACCF/AHA stage. In some embodiments, the method reduces a patient's ACCF/AHA stage from Stage D to Stage C. In some embodiments, the method reduces a patient's ACCF/AHA stage from Stage D to Stage B. In some embodiments, the method reduces a patient's ACCF/AHA stage from Stage D to Stage A. In some embodiments, the method reduces a patient's ACCF/AHA stage from Stage C to Stage B. In some embodiments, the method reduces a patient's ACCF/AHA stage from Stage C to Stage A. In some embodiments, the method reduces a patient's ACCF/AHA stage or from Stage B to Stage A.

In some embodiments of the present disclosure, a patient has Killip Classification of HF complicating AMI Class I heart failure. In some embodiments, a patient has Killip Classification of HF complicating AMI Class II heart failure. In some embodiments, a patient has Killip Classification of HF complicating AMI Class III heart failure. In some embodiments, a patient has or Killip Classification of HF complicating AMI Class IV heart failure.

In some embodiments, methods of the present disclosure reduce a patient's Killip Classification of HF complicating AMI class. In some embodiments, the method reduces a patient's Killip Class from Class IV to Class III. In some embodiments, the method reduces a patient's Killip Class from Class IV to Class II. In some embodiments, the method reduces a patient's Killip Class from Class IV to Class I. In some embodiments, the method reduces a patient's Killip Class from Class III to Class II. In some embodiments, the method reduces a patient's Killip Class from Class III to Class I. In some embodiments, the method reduces a patient's Killip Class or from Class II to Class I.

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

In some embodiments of the present disclosure, a patient has one or more minor Framingham criteria for diagnosis of HF. In some embodiments, a patient has one or more conditions selected from the group consisting of bilateral ankle edema, nocturnal cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion, decrease in vital capacity by ⅓ from maximum recorded, and tachycardia (heart rate greater than 120/min).

In some embodiments of the present disclosure, a patient has at least two Framingham major criteria. In some embodiments, a patient has at least one major Framingham criteria and at least two minor Framingham criteria.

In some embodiments, methods of the present disclosure reduce the number of Framingham criteria for heart failure that a patient has. In some embodiments, the method decreases the number of major Framingham criteria for heart failure that a patient has. In some embodiments, the method decreases the number of minor Framingham criteria for heart failure that a patient has.

In some embodiments of the present disclosure, a 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, a patient has one or more symptoms selected from the group consisting of breathlessness, orthopnea, paroxysmal nocturnal dyspnea, reduced exercise tolerance, fatigue, tiredness, increased time to recover after exercise, and ankle swelling. In some embodiments, a patient has one or more less typical symptoms selected from the group consisting of nocturnal cough, wheezing, bloated feeling, loss of appetite, confusion (especially in the elderly), depression, palpitations, dizziness, syncope, and bendopnea.

In some embodiments of the present disclosure, a patient has one or more signs of HF. In some embodiments, a patient has one or more signs of HF selected from the group consisting of elevated jugular venous pressure, hepatojugular reflux, third heart sound (gallop rhythm), laterally displaced apical impulse. In some embodiments, a patient has one or more less specific signs of HF. In some embodiments, a patient has one or more less specific signs of HF. In some embodiments, a patient has one or more less specific signs of HF selected from the group consisting of weight gain (>2 kg/week), weight loss (in advanced HF), tissue wasting (cachexia), cardiac murmur, peripheral edema (ankle, sacral, scrotal), pulmonary crepitations, reduced air entry and dullness to percussion at lung bases (pleural effusion), tachycardia, irregular pulse, tachypnoea, Cheyne Stokes respiration, hepatomegaly, ascites, cold extremities, oliguria, and narrow pulse pressure.

In some embodiments, methods of the present disclosure reduce the number of signs and/or symptoms of heart failure that a patient has. In some embodiments, the method decreases the number of signs of heart failure that a patient has. In some embodiments, the method decreases the number of symptoms of heart failure that a patient has.

In some embodiments of the present disclosure, a patient has elevated brain natriuretic peptide (BNP) levels as compared to a healthy patient. In some embodiments, a 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, methods of the present disclosure decrease BNP levels in a patient 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, methods of the present disclosure decrease BNP levels in a patient by at least 5 pg/mL (e.g., 5, 10, 50, 100, 200, 500, 1000, or 5000 pg/mL). In some embodiments, methods of the present disclosure decrease BNP levels to normal levels (i.e., <100 pg/ml).

In some embodiments of the present disclosure, a patient has elevated N-terminal pro-BNP (NT-proBNP) levels as compared to a healthy patient. In some embodiments, a 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, methods of the present disclosure decrease NT-proBNP levels in a patient 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, methods of the present disclosure decrease NT-proBNP levels in a patient 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, methods of the present disclosure decrease NT-proBNP levels to normal levels (i.e., <100 pg/ml).

In some embodiments of the present disclosure, a patient has elevated troponin levels as compared to a healthy patient. In some embodiments, methods of the present disclosure decrease troponin levels in a patient 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%).

In some embodiments, methods of the present disclosure decrease left ventricular hypertrophy in a patient. In some embodiments, methods of the present disclosure decrease left ventricular hypertrophy in a patient by at least 1% (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%). In some embodiments, methods of the present disclosure reduce a patient's hospitalization rate by at least 1% (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 6%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, methods of the present disclosure reduce a patient's rate of worsening of heart failure by at least 1% (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 6%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%).

In some embodiments of the present disclosure, a patient has diastolic dysfunction of the left ventricle (LV). In some embodiments, a patient has systolic dysfunction of the left ventricle (LV). In some embodiments, methods of the present disclosure increase a patient's LV diastolic function by at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 7%, 80%, 85%, 90%, 95%, or 100%).

In some embodiments of the present disclosure, a patient has an ejection fraction of less than 45% (e.g., 10, 15, 20, 25, 30, 35, 40, or 45%). In some embodiments, methods of the present disclosure increase ejection fraction to normal levels (i.e., >45%).

In some embodiments, methods of the present disclosure increase a patient's cardiac output 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, methods of the present disclosure increase ejection fraction in a patient 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%).

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

In some embodiments, methods of the present disclosure reduce a patient's Borg dyspnea index (BDI). In some embodiments, methods of the present disclosure reduce a patient's BDI by at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 index points).

In some embodiments of the disclosure, a patient is assessed for heart failure using echocardiography. In some embodiments, a patient is assessed for heart failure using cardiac magnetic resonance imaging (CMR). In some embodiments, a patient is assess for heart failure using CMR with late gadolinium enhancement (LGE). In some embodiments, a patient is assessed for one or more of 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 equation FS=100%×[(EDD−ESD)/EDD]), LV end diastolic volume (LVEDV), LV end systolic volume (LVESV), ejection fraction (calculated using the equation 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, heart failure in a patient is assessed using cardiac imaging selected from the group consisting of multigated acquisition (MUGA), Chest X-Ray, single-photon emission computed tomography (SPECT) and radionucleotide ventriculography, positron emission tomography (PET), coronary angiography, and cardiac computing tomography (CT).

In some embodiments, methods of the present disclosure further comprise administering to a patient an additional supportive therapy or active agent. In some embodiments, the additional supportive therapy or active agent is selected from the group consisting of angiotensin-converting enzyme (ACE) inhibitors, beta blockers, angiotensin II receptor blockers (ARB), mineralcorticoid/aldosterone receptor antagonists (MRAs), glucocorticoids, statins, Sodium-glucose co-transporter 2 (SGLT2) inhibitors, an implantable cardioverter defibrillator (ICD), angiotensin receptor neprilysin inhibitors (ARNI), and diuretics. In some embodiments, the additional active agent and/or supportive therapy is selected from the group consisting of: benazepril, captopril, enalapril, lisinopril, perindopril, ramipril (e.g., ramipen), trandolapril, zofenopril, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, timolol; losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan, azilsartan, salprisartan, telmisartan, progesterone, eplerenone and spironolactone, beclomethasone, betamethasone, budesonide, cortisone, deflazacort, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, methylprednisone, prednisone, triamcinolone, finerenone, atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pravastatin (Pravachol), pitavastatin (Livalo), simvastatin (Zocor), rosuvastatin (Crestor), canagliflozin, dapagliflozin (e.g., Farxiga), empagliflozin, valsartan and sacubitril (a neprilysin inhibitor), furosemide, bumetanide, torasemide, bendroflumethiazide, hydrochlorothiazide, metolazone, indapamidec, spironolactone/eplerenone, amiloride triamterene, hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), and I_f-channel inhibitor (e.g., Ivabradine).

In some embodiments of the present disclosure, a patient has a comorbidity selected from the group consisting of advanced age, anemia, arterial hypertension, atrial fibrillation, cognitive dysfunction, diabetes, hypercholesterolemia, iron deficiency, kidney dysfunction, metabolic syndrome, obesity, physical deconditioning, potassium disorders, pulmonary disease (e.g., COPD), and sleep apnea.

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

In some embodiments of the present disclosure, an 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 an amino acid sequence that begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 366 and ends 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, 131, 132, 133, 134, or 135 of SEQ ID NO: 366.

In some embodiments of the present disclosure, an 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 an amino acid sequence that begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 366 and ends 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, 131, 132, 133, 134, or 135 of SEQ ID NO: 367.

In some embodiments of the present disclosure, an 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 an amino acid sequence that begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of SEQ ID NO: 366 and ends 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, 131, 132, 133, 134, or 135 of SEQ ID NO: 368.

In some embodiments of the present disclosure, an ActRIIA polypeptide is a fusion polypeptide comprising an ActRIIA polypeptide domain and one or more heterologous domains. In some embodiments, an ActRIIA polypeptide is an ActRIIA-Fc fusion polypeptide. In some embodiments, the fusion polypeptide further comprises a linker domain positioned between the ActRIIA polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, a linker domain is selected from: TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.

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.

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.

In some embodiments of the present disclosure, an ActRII-ALK4 antagonist is a homodimer polypeptide. In some embodiments, an ActRII-ALK4 antagonist is a heteromultimer polypeptide. In some embodiments, a heteromultimer polypeptide comprises an ActRIIA polypeptide and an ALK4 polypeptide. In some embodiments, the heteromultimer polypeptide comprises an ActRIIA polypeptide and an ALK7 polypeptide.

In some embodiments of the present disclosure, an 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, 422.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ALK4 polypeptide is a fusion polypeptide comprising an ALK4 polypeptide domain and one or more heterologous domains. In some embodiments, an ALK7 polypeptide is a fusion polypeptide comprising an ALK7 polypeptide domain and one or more heterologous domains. In some embodiments, an ALK4 polypeptide is an ALK4-Fc fusion polypeptide. In some embodiments, an ALK7 polypeptide is an ALK7-Fc fusion polypeptide. In some embodiments, the ALK4-Fc fusion polypeptide further comprises a linker domain positioned between the ALK4 polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, the ALK7-Fc fusion polypeptide further comprises a linker domain positioned between the ALK7 polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, the linker domain is selected from: TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a) 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: 13, 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: 13; b) 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: 14, 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: 14; c.) 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: 15, 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: 15; d.) 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: 16, 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: 16; and e.) 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: 17, 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: 17.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 13, 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: 13; b.) 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: 14, 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: 14; c.) 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: 15, 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: 15; d.) 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: 16, 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: 16; and e.) 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: 17, 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: 17.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 18, 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: 19; and b.) 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: 19, 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: 18.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 18, 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: 19; and b.) 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: 19, 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: 18.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 20, 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: 21; and b.) 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: 21, 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: 20.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 20, 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: 21; and b.) 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: 21, 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: 20.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 22, 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; and b.) 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, 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: 22.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 22, 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; and b.) 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, 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: 22.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 24, 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: 25; and b.) 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: 25, 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: 24.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 24, 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: 25; and b.) 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: 25, 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: 24.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 26, 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: 27; and b.) 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: 27, 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: 26.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 26, 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: 27; and b.) 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: 27, 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: 26.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ALK4-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an ALK7-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ALK4-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ALK7-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ALK4-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ActRIIA-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an ALK7-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ActRIIA-Fc fusion polypeptide Fc domain comprises 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.

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

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

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 an amino acid sequence that begins at any one of amino acids 20-29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acids 109-134 (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.

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 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 25-131 of SEQ ID NO: 2.

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 20-134 of SEQ ID NO: 2.

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 the amino acid sequence of SEQ ID NO: 53.

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 the amino acid sequence of SEQ ID NO: 388.

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 the amino acid sequence of SEQ ID NO: 389.

In some embodiments of the present disclosure, an ActRIIB polypeptide is a fusion polypeptide comprising an ActRIIB polypeptide domain and one or more heterologous domains. In some embodiments, an ActRIIB polypeptide is an ActRIIB-Fc fusion polypeptide. In some embodiments, the fusion polypeptide further comprises a linker domain positioned between the ActRIIB polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, the linker domain is selected from: TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS. 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.

In some embodiments of the present disclosure, an ActRIIB polypeptide comprises one or more amino acid substitution with respect to the amino acid sequence of 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. In some embodiments, an ActRIIB polypeptide comprises one or more amino acid substitution 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.

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

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 of SEQ ID NO: 2 and an R substitution at the position corresponding to N83.

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 of SEQ ID NO: 2 and an R substitution at the position corresponding to N83.

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 of SEQ ID NO: 2 and an R substitution at the position corresponding to N83.

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 the position corresponding to F82 of SEQ ID NO: 2.

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: 339. 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: 341. In some embodiments, the polypeptide comprises an H substitution at the position corresponding to L79 of SEQ ID NO: 2 and an I substitution at the position corresponding to F82.

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 the position corresponding to L79 of SEQ ID NO: 2.

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 the position corresponding to L79 of SEQ ID NO: 2 and a K substitution at the position corresponding to F82.

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 the position corresponding to E50 of SEQ ID NO: 2.

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 the position corresponding to L38 of SEQ ID NO: 2 and an R substitution at the position corresponding to L79.

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 an G substitution at the position corresponding to V99 of SEQ ID NO: 2.

In some embodiments of the present disclosure, an ActRIIB polypeptide is a homodimer polypeptide. In some embodiments, an ActRIIB polypeptide is a heterodimer polypeptide.

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 an amino acid sequence that begins at any one of amino acids 20-29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acids 109-134 (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 one or more amino acid substitutions at a position of 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.

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 an amino acid sequence that begins at any one of amino acids 20-29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acids 109-134 (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 one or more amino acid substitutions at a position 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.

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, 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: 389. In some embodiments, an ActRIIB polypeptide comprises one or more amino acid substitution 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.

In some embodiments of the present disclosure, a heteromultimer polypeptide comprises an ActRIIA polypeptide and an ALK4 polypeptide. In some embodiments, a heteromultimer polypeptide comprises an ActRIIA polypeptide and an ALK7 polypeptide. In some embodiments, an 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, 422. In some embodiments, an 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.

In some embodiments of the present disclosure, an ActRIIB polypeptide is a fusion polypeptide comprising an ActRIIB polypeptide domain and one or more heterologous domains. In some embodiments, an ALK4 polypeptide is a fusion polypeptide comprising an ALK4 polypeptide domain and one or more heterologous domains. In some embodiments, an ALK7 polypeptide is a fusion polypeptide comprising an ALK7 polypeptide domain and one or more heterologous domains. In some embodiments, an ActRIIB polypeptide is an ActRIIB-Fc fusion polypeptide. In some embodiments, an ALK4 polypeptide is an ALK4-Fc fusion polypeptide. In some embodiments, an ALK7 polypeptide is an ALK7-Fc fusion polypeptide. In some embodiments, an ActRIIB-Fc fusion polypeptide further comprises a linker domain positioned between the ActRIIB polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, the ALK4-Fc fusion polypeptide further comprises a linker domain positioned between the ALK4 polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, the ALK7-Fc fusion polypeptide further comprises a linker domain positioned between the ALK7 polypeptide domain and the one or more heterologous domains or Fc domain. In some embodiments, the linker domain is selected from: TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 13, 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: 13; b.) 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: 14, 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: 14; c.) 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: 15, 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: 15; d.) 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: 16, 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: 16; and e.) 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: 17, 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: 17.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 13, 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: 13; b.) 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: 14, 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: 14; c.) 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: 15, 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: 15; d.) 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: 16, 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: 16; and e.) 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: 17, 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: 17.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 18, 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: 19; and b) 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: 19, 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: 18.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 18, 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: 19; and b.) 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: 19, 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: 18.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 20, 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: 21; and b.) 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: 21, 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: 20.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 20, 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: 21; and b.) 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: 21, 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: 20.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 22, 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; and b.) 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, 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: 22.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 22, 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; and b.) 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, 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: 22.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 24, 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: 25; and b.) 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: 25, 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: 24.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 24, 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: 25; and b.) 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: 25, 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: 24.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 26, 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: 27; and b.) 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: 27, 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: 26.

In some embodiments of the present disclosure, a heteromultimer comprises an Fc domain selected from: a.) 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: 26, 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: 27; and b.) 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: 27, 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: 26.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ALK4-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an ALK7-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and a aspartic acid at amino acid position 217, and wherein 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ALK4-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ALK7-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ALK4-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ActRIIB-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an ALK7-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, a tryptophan at amino acid position 144, and a arginine at amino acid position 435, and wherein the ActRIIB-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments of the present disclosure, an ActRII-ALK4 antagonist is a follistatin polypeptide. In some embodiments, the follistatin polypeptide 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: 390, 391, 392, 393, and 394.

In some embodiments of the present disclosure, an 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.

In some embodiments of the present disclosure, an ActRII-ALK4 antagonist is an antibody or 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 bi-specific antibody.

In some embodiments of the present disclosure, an ActRII-ALK4 antagonist is a small molecule or combination of small molecules. In some embodiments, the small molecule or combination of small molecules inhibits 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 of the present disclosure, an ActRII-ALK4 antagonist is a polynucleotide or 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 DESCRIPTION OF THE DRAWING

FIG. 1 shows an alignment of extracellular domains of human ActRIIB (SEQ ID NO: 1) and human ActRIIA (SEQ ID NO: 367) with the residues that are deduced herein, based on composite analysis of multiple ActRIIB and ActRIIA crystal structures, to directly contact ligand indicated with boxes.

FIG. 2 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 the 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 for the variants described herein are based on the numbering in SEQ ID NO: 2 FIG. 3 shows the amino acid sequence of a human ActRIIB extracellular domain polypeptide (SEQ ID NO: 1).

FIG. 4 shows a nucleic acid sequence encoding human ActRIIB precursor polypeptide. SEQ ID NO: 4 consists of nucleotides 434-1972 of NCBI Reference Sequence NM_001106.4.

FIG. 5 shows a nucleic acid sequence (SEQ ID NO: 3) encoding a human ActRIIB(20-134) extracellular domain polypeptide.

FIG. 6 shows a multiple sequence alignment of various vertebrate ActRIIB precursor polypeptides without their intracellular domains (SEQ ID NOs: 358-363), human ActRIIA precursor polypeptide without its intracellular domain (SEQ ID NO: 364), and a consensus ActRII precursor polypeptide (SEQ ID NO: 365). Upper case letters in the consensus sequence indicate positions that are conserved. Lower case letters in the consensus sequence indicate an amino acid residue that is the predominant form, but not universal at that position.

FIG. 7 shows multiple sequence alignment of Fc domains from human IgG isotypes using Clustal 2.1. Hinge regions are indicated by dotted underline. Double underline indicates examples of positions engineered in IgG1 (SEQ ID NO: 13) Fc to promote asymmetric chain pairing and the corresponding positions with respect to other isotypes IgG4 (SEQ ID NO: 17), IgG2 (SEQ ID NO: 14), and IgG3 (SEQ ID NO: 15).

FIG. 8A and FIG. 8B show schematic examples of heteromeric polypeptide complexes comprising a variant ActRIIB polypeptide (indicated as “X”) and either an ALK4 polypeptide (indicated as “Y”) or an ALK7 polypeptide (indicated as “Y”). In the illustrated embodiments, the variant ActRIIB polypeptide is part of a fusion polypeptide that comprises a first member of an interaction pair (“C₁”), and either an ALK4 polypeptide or an ALK7 polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C₂”). Suitable interaction pairs include, for example, heavy chain 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 positioned between the variant ActRIIB polypeptide, ALK4 polypeptide, or ALK7 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 may associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See FIG. 8A. Alternatively, the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair associate preferentially with each other rather than self-associate. See FIG. 8B.

FIG. 9 shows a multiple sequence alignment of various vertebrate ALK4 proteins and human ALK4 (SEQ ID NOs: 414-420).

FIG. 10 shows a multiple sequence alignment of various vertebrate ActRIIA proteins and human ActRIIA (SEQ ID NOs: 367, 371-377).

FIGS. 11A and 11B show two schematic examples of heteromeric protein complexes comprising type I receptor and type II receptor polypeptides. FIG. 11A depicts a heterodimeric protein complex comprising one type I receptor fusion polypeptide and one type II receptor fusion polypeptide, which can be assembled covalently or noncovalently via a multimerization domain contained within each polypeptide chain. Two assembled multimerization domains constitute an interaction pair, which can be either guided or unguided. FIG. 11B depicts a heterotetrameric protein complex comprising two heterodimeric complexes as depicted in FIG. 11A. Complexes of higher order can be envisioned.

FIG. 12 show a schematic example of a heteromeric protein complex comprising a type I receptor polypeptide (indicated 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 an extracellular domain of an ALK4 protein from humans or other species such as those described herein) and a type II receptor polypeptide (indicated 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 an extracellular domain of an ActRIIB protein from humans or other species as such as those described herein). In the illustrated embodiments, the type I receptor polypeptide is part of a fusion polypeptide that comprises a first member of an interaction pair (“C₁”), and the type II receptor polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C₂”). In each fusion polypeptide, a linker may be positioned 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 may be a guided (asymmetric) pair, meaning that the members of the pair associate preferentially with each other rather than self-associate, or the interaction pair may be unguided, meaning that the members of the pair may associate with each other or self-associate without substantial preference and may have the same or different amino acid sequences. Traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, whereas a variety of engineered Fc domains have been designed as guided (asymmetric) interaction pairs [e.g., Spiess et al (2015) Molecular Immunology 67(2A): 95-106].

FIGS. 13A-13D show schematic examples of heteromeric protein complexes 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 an extracellular domain of an ALK4 protein from humans 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 an extracellular domain of an ActRIIB protein from humans or other species such as those described herein). In the illustrated embodiments, the ALK4 polypeptide is part of a fusion polypeptide that comprises a first member of an interaction pair (“C₁”), and the ActRIIB polypeptide is part of a fusion polypeptide that comprises a second member of an interaction pair (“C₂”). Suitable interaction pairs included, for example, heavy chain 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 positioned 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 may associate with each other or self-associate without substantial preference, and they may have the same or different amino acid sequences. See FIG. 13A. Alternatively, the interaction pair may be a guided (asymmetric) pair, meaning that the members of the pair associate preferentially with each other rather than self-associate. See FIG. 13B. Complexes of higher order can be envisioned. See FIGS. 13C and 13D.

FIG. 14 shows the purification of ActRIIA-hFc expressed in CHO cells. The protein purifies as a single, well-defined peak as visualized by sizing column (top panel) and Coomassie stained SDS-PAGE (bottom panel) (left lane: molecular weight standards; right lane: ActRIIA-hFc).

FIG. 15 shows the binding of ActRIIA-hFc to activin (top panel) and GDF-11 (bottom panel), as measured by Biacore™ assay.

FIG. 16A and FIG. 16B show values for ligand binding kinetics of homodimeric Fc-fusion polypeptides comprising 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 that the value is not detectable over concentration range tested. Transient* indicates that the value is indeterminate due to transient nature of interaction. Control sample is ActRIIB-G1Fc (SEQ ID NO: 5).

FIG. 17 shows values for ligand binding kinetics of homodimeric Fc-fusion polypeptides comprising 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 that the value is not detectable over concentration range tested. Transient binding* indicates that the value is indeterminate due to transient nature of interaction. Control sample is ActRIIB-G1Fc (SEQ ID NO: 5).

FIG. 18 shows values for ligand binding kinetics of homodimeric Fc-fusion polypeptides comprising variant or unmodified ActRIIB domains, as determined by surface plasmon resonance at 25° C. ND #indicates that the value is not detectable over concentration range tested. Amino acid numbering is based on SEQ ID NO: 2.

FIG. 19 shows comparative ligand binding data for an ALK4-Fc:ActRIIB-Fc heterodimeric protein complex compared to ActRIIB-Fc homodimer and ALK4-Fc homodimer. For each protein complex, ligands are ranked by k_off, a kinetic constant that correlates well with ligand signaling inhibition, and listed in descending order of binding affinity (ligands bound most tightly are listed at the top). At left, yellow, red, green, and blue lines indicate magnitude of the off-rate constant. Solid black lines indicate ligands whose binding to heterodimer is enhanced or unchanged compared with homodimer, whereas dashed red lines indicate substantially reduced binding compared with homodimer. As shown, the ActRIIB-Fc:ALK4-Fc heterodimer displays enhanced binding to activin B compared with either homodimer, retains strong binding to activin A, GDF8, and GDF11 as observed with ActRIIB-Fc homodimer, and exhibits substantially reduced binding to BMP9, BMP10, and GDF3. Like ActRIIB-Fc homodimer, the heterodimer retains intermediate-level binding to BMP6.

FIG. 20 shows comparative ActRIIB-Fc:ALK4-Fc heterodimer/ActRIIB-Fc:ActRIIB-Fc homodimer IC₅₀ data as determined by an A-204 Reporter Gene Assay as described herein. ActRIIB-Fc:ALK4-Fc heterodimer inhibits activin A, activin B, GDF8, and GDF11 signaling pathways similarly to the ActRIIB-Fc:ActRIIB-Fc homodimer. However, ActRIIB-Fc:ALK4-Fc heterodimer inhibition of BMP9 and BMP10 signaling pathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fc homodimer. These data demonstrate that ActRIIB:ALK4 heterodimers are more selective antagonists of activin A, activin B, GDF8, and GDF11 compared to corresponding ActRIIB:ActRIIB homodimers.

FIG. 21 shows comparative ligand binding data for an ActRIIB-Fc:ALK7-Fc heterodimeric protein complex compared to ActRIIB-Fc homodimer and ALK7-Fc homodimer. For each protein complex, ligands are ranked by k_off, a kinetic constant that correlates well with ligand signaling inhibition, and listed in descending order of binding affinity (ligands bound most tightly are listed at the top). At left, yellow, red, green, and blue lines indicate magnitude of the off-rate constant. Solid black lines indicate ligands whose binding to heterodimer is enhanced or unchanged compared with homodimer, whereas dashed red lines indicate substantially reduced binding compared with homodimer. As shown, four of the five ligands with strong binding to ActRIIB-Fc homodimer (activin A, BMP10, GDF8, and GDF11) exhibit reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, the exception being activin B which retains tight binding to the heterodimer. Similarly, three of four ligands with intermediate binding to ActRIIB-Fc homodimer (GDF3, BMP6, and particularly BMP9) exhibit reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, whereas binding to activin AC is increased to become the second strongest ligand interaction with the heterodimer overall. Finally, activin C and BMP5 unexpectedly bind the ActRIIB-Fc:ALK7 heterodimer with intermediate strength despite no binding (activin C) or weak binding (BMP5) to ActRIIB-Fc homodimer. No ligands tested bind to ALK7-Fc homodimer.

FIG. 22 shows a multiple sequence alignment of ALK7 extracellular domains derived from various vertebrate species (SEQ ID NOs: 425-430).

FIG. 23 ActRIIB-Fc:ALK4-Fc corrected left ventricular structural alterations during left heart remodeling. A. Illustration of left heart remodeling with dilated cardiomyopathy. Modified figure from Houser et al., 2012. FIGS. B-E show results of Mid-age Mdx-Vehicle mice who received an iso-volume amount of PBS vehicle for 6 months and Old Mdx-Vehicle mice who received iso-volume amount of PBS vehicle for 2 months, or Mid-age Mdx-ActRIIB-Fc:ALK4-Fc mice who received ActRIIB-Fc:ALK4-Fc (10 mg/kg) for 6 months and Old Mdx-ActRIIB-Fc:ALK4-Fc who received ActRIIB-Fc:ALK4-Fc (10 mg/kg) for 2 months. Figure B shows that LVESV was increased in Mid-age Mdx-Vehicle (n=8) mice compared to either Mid-age WT-Vehicle (n=7, p>0.05) or Young W T mice (n=3, p>0.05), but as shown by Mid-age Mdx-ActRIIB-Fc:ALK4-Fc, LVESV was significantly reduced by ActRIIB-Fc:ALK4-Fc treatment (n=6, p<0.5). LVESV was increased in Old Mdx-Vehicle (n=3) mice compared to either Old WT-Vehicle (n=5, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Old-age Mdx-ActRIIB-Fc:ALK4-Fc, LVESV was significantly reduced by ActRIIB-Fc:ALK4-Fc treatment (n=4, p<0.001). C. LV eccentric hypertrophy references to the ratio of mass (LVM) to volume (LVESV) and is described as hypertrophy index. Hypertrophy index was reduced in Mid-age Mdx-Vehicle (n=8) mice compared to either Mid-age WT-Vehicle (n=7, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Mid-age Mdx-ActRIIB-Fc:ALK4-Fc, hypertrophy index was increased by ActRIIB-Fc:ALK4-Fc treatment (n=6, p>0.5). Hypertrophy index was decreased in Old Mdx-Vehicle mice (n=3) mice compared to either Old WT-Vehicle (n=5, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Old-age Mdx-ActRIIB-Fc:ALK4-Fc, hypertrophy index was increased by ActRIIB-Fc:ALK4-Fc treatment (n=4, p>0.05). D. LV relative wall thickness as referenced to the ratio of LV wall thickness (LVWT) to LV diameter at the end of systole (LVESD). Relative wall thickness was reduced in Mid-age Mdx-Vehicle (n=8) mice compared to either Mid-age WT-Vehicle (n=7, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Mid-age Mdx-ActRIIB-Fc:ALK4-Fc, relative wall thickness was increased by ActRIIB-Fc:ALK4-Fc treatment (n=6, p>0.5). Relative wall thickness was decreased in Old Mdx-Vehicle (n=3) mice compared to either Old WT-Vehicle (n=5, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Old Mdx-ActRIIB-Fc:ALK4-Fc, relative wall thickness was increased by ActRIIB-Fc:ALK4-Fc treatment (n=4, p>0.05). E. Normalized heart weight references to the ratio of whole heart weight to body weight. Normalized heart weight was increased in Old Mdx-Vehicle (n=3) mice compared to either Old WT-Vehicle (n=5, p>0.05) or Young WT mice (n=3, p<0.01), but as shown by Old Mdx-ActRIIB-Fc:ALK4-Fc, normalized heart weight was reduced by ActRIIB-Fc:ALK4-Fc treatment (n=4, p>0.05). Normalized heart weight was significantly increased in Old WT-Vehicle (n=5) mice compared to Young WT mice (n=3, p<0.05).

FIG. 24 ActRIIB-Fc:ALK4-Fc rescued left ventricular systolic dysfunction during left heart remodeling. Figures A-D show results of Mid-age Mdx-Vehicle mice who received iso-volume amount of PBS vehicle for 6 months and Old Mdx-Vehicle mice who received iso-volume amount of PBS vehicle for 2 months, or Mid-age Mdx-ActRIIB-Fc:ALK4-Fc mice who received ActRIIB-Fc:ALK4-Fc (10 mg/kg) for 6 months and Old Mdx-ActRIIB-Fc:ALK4-Fc who received ActRIIB-Fc:ALK4-Fc (10 mg/kg) for 2 months. A. Ejection fraction was reduced in Mid-age Mdx-Vehicle (n=8) mice compared to either Mid-Age WT-Vehicle (n=7, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Mid-age Mdx-ActRIIB-Fc:ALK4-Fc, ejection fraction was significantly increased by ActRIIB-Fc:ALK4-Fc treatment (n=6, p<0.5). Ejection fraction was reduced in Old Mdx-Vehicle (n=3) mice compared to either Old WT-Vehicle (n=5, p>0.05) or Young-WT mice (n=3, p>0.05), but as shown by Old Mdx-ActRIIB-Fc:ALK4-Fc, ejection fraction was significantly increased by ActRIIB-Fc:ALK4-Fc treatment (n=4, p<0.01). B. Fractional shortening was reduced in Mid-age Mdx-Vehicle (n=8) mice compared to either Mid-age WT-Vehicle (n=7, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Mid-age Mdx-ActRIIB-Fc:ALK4-Fc, fractional shortening was significantly increased by ActRIIB-Fc:ALK4-Fc treatment (n=6, p<0.5). Fractional shortening was reduced in Old Mdx-Vehicle (n=3) mice compared to either Old WT-Vehicle (n=5, p>0.05) or Young WT mice (n=3, p>0.05), but as shown by Old Mdx-ActRIIB-Fc:ALK4-Fc, fractional shortening was significantly increased by ActRIIB-Fc:ALK4-Fc treatment (n=4, p<0.01). C. Serum cTnI level was measured by a high sensitivity mouse cTnI ELISA kit. Serum cTnI level was dramatically increased in Mid-age Mdx-Vehicle (n=3) mice compared to either Mid-age WT-Vehicle (n=6, p<0.05) or Young WT mice (n=2, p<0.05), but as shown by Mid-age Mdx-ActRIIB-Fc:ALK4-Fc, serum cTnI level was significantly reduced by ActRIIB-Fc:ALK4-Fc treatment (n=5, p<0.05). D. A moderate inverse correlation (Pearson's coefficient, r=−0.39) was present between ejection fraction and serum cTnI of the data presented in FIGS. 2A and 2C (p<0.01).

DETAILED DESCRIPTION

1. Overview

In certain aspects, the disclosure relates to methods of using TGF-β superfamily ligand antagonists, in particular ActRII-ALK4 antagonists, to treat heart failure. For example, ActRII-ALK4 antagonists as described herein may be used to treat, prevent, or reduce the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure.

Heart Failure (HF) is a clinical syndrome characterized by symptoms that include breathlessness, ankle swelling and fatigue, that may be accompanied by signs that include elevated jugular venous pressure, pulmonary crackles and peripheral edema caused by a structural and/or functional cardiac abnormality. HF typically results in a reduced cardiac output and/or elevated intracardiac pressure at rest or during stress.

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

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

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

Two related type II receptors, ActRIIA and ActRIIB, have been identified as the type II receptors for activins (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins, 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). Applicants have found that soluble ActRIIA-Fc fusion polypeptides and ActRIIB-Fc fusion polypeptides have substantially different effects in vivo, with ActRIIA-Fc having primary effects on bone and ActRIIB-Fc having primary effects on skeletal muscle.

Ligands of the TGF-beta superfamily share the same dimeric structure in which the central 3-½ turn helix of one monomer packs against the concave surface formed by the beta-strands of the other monomer. The majority of TGF-beta family members are further stabilized by an intermolecular disulfide bond. This disulfide bonds traverses through a ring formed by two other disulfide bonds generating what has been termed a ‘cysteine knot’ motif [Lin et al. (2006) Reproduction 132: 179-190; and Hinck et al. (2012) FEBS Letters 586: 1860-1870].

Activins are members of the TGF-beta superfamily and were initially discovered as regulators of secretion of follicle-stimulating hormone, but subsequently various reproductive and non-reproductive roles have been characterized. There are three principal activin forms (A, B, and AB) that are homo/heterodimers of two closely related β subunits (β_Aβ_A, β_Bβ_B, and β_Aβ_B, respectively). The human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing β_C or β_E are also known. In the TGF-beta superfamily, activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and 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 several tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, in the regulation of follicle-stimulating hormone (FSH) secretion from the pituitary, activin promotes FSH synthesis and secretion, while inhibin reduces FSH synthesis and secretion. Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS) and α₂-macroglobulin.

As described herein, agents that bind to “activin A” are agents that specifically bind to the β_A subunit, whether in the context of an isolated β_A subunit or as a dimeric complex (e.g., a β_Aβ_A homodimer or a β_Aβ_B heterodimer). In the case of a heterodimer complex (e.g., a β_Aβ_B heterodimer), agents that bind to “activin A” are specific for epitopes present within the RA subunit, but do not bind to epitopes present within the non-β_A subunit of the complex (e.g., the β_B subunit of the complex). Similarly, agents disclosed herein that antagonize (inhibit) “activin A” are agents that inhibit one or more activities as mediated by a β_A subunit, whether in the context of an isolated β_A subunit or as a dimeric complex (e.g., a β_Aβ_A homodimer or a β_Aβ_B heterodimer). In the case of β_Aβ_B heterodimers, agents that inhibit “activin A” are agents that specifically inhibit one or more activities of the β_A subunit, but do not inhibit the activity of the non-β_A subunit of the complex (e.g., the β_B subunit of the complex). This principle applies also 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 inhibit one or more activities as mediated by the β_A subunit and one or more activities as mediated by the β_B subunit.

The BMPs and GDFs together form a family of cysteine-knot cytokines sharing the characteristic fold of the TGF-beta 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. Besides the ability to induce bone formation, which gave the BMPs their name, the BMP/GDFs display morphogenetic activities in the development of a wide range of tissues. BMP/GDF homo- and hetero-dimers interact with combinations of type I and type II receptor dimers to produce multiple possible signaling complexes, leading to the activation of one of two competing sets of SMAD transcription factors. BMP/GDFs have highly specific and localized functions. These are regulated in a number of ways, including the developmental restriction of BMP/GDF expression and through the secretion of several specific BMP antagonist proteins that bind with high affinity to the cytokines. Curiously, a number of these antagonists resemble TGF-beta superfamily ligands.

Growth and differentiation factor-8 (GDF8) is also known as myostatin. GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in the developing and adult skeletal muscle. The GDF8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the skeletal muscle (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, strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8). Studies have also shown that muscle wasting associated with HIV-infection in humans is accompanied by increases in GDF8 polypeptide expression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). In addition, GDF8 can modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell 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 which bind to GDF8 or structurally related polypeptides and inhibit their biological activity include follistatin, and potentially, follistatin-related polypeptides (Gamer et al. (1999) Dev. Biol., 208: 222-232).

Growth and 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, maxillary and mandibular arches, and dorsal root ganglia during mouse development (Nakashima et al., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role in patterning both mesodermal and neural tissues (Gamer et al., 1999, Dev Biol., 208:222-32). GDF11 was shown to be a negative regulator of chondrogenesis and myogenesis in developing chick limb (Gamer et al., 2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle also suggests its role in regulating muscle growth in a similar way to GDF8. In addition, the expression of GDF11 in brain suggests that GDF11 may also possess activities that relate to the function of the nervous system. Interestingly, GDF11 was found to inhibit neurogenesis in the olfactory epithelium (Wu et al., 2003, Neuron. 37:197-207).

In part, the examples of the disclosure demonstrate that an ActRIIB:ALK4 heterodimer is effective to ameliorate various morphological and functional deficits during left heart remodeling in a murine model of HFrEF (Mdx model). In particular, LV end systolic diameter was significantly reduced in ActRIIB:ALK4 heterodimer treated mice compared to untreated groups, indicating that ActRIIB:ALK4 heterodimer improved LV contractility. The data further suggest that, in addition to ActRIIB:ALK4 heteromultimers, other ActRII-ALK4 antagonists may be useful in treating heart failure.

In certain aspects, an ActRII-ALK4 antagonist to be 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 (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure) is an ActRII-ALK4 ligand trap polypeptide antagonist including variants thereof as well as 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, that are capable of binding to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and/or BMP10). Therefore, an ActRII-ALK4 ligand trap generally includes polypeptides that are 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, as well has homomultimers (e.g., ActRIIB homodimers) and heteromultimers (e.g., ActRIIB-ALK4 or ActRIIB-ALK7 heterodimers). In some embodiments, an ActRII-ALK4 ligand trap comprises an ActRIIA polypeptide, including variants thereof, as well has homomultimers (e.g., ActRIIA homodimers) and heteromultimers (e.g., ActRIIA-ALK4 or ActRIIA-ALK7 heterodimers). In other embodiments, an ActRII-ALK ligand trap comprises a soluble ligand trap protein including, but not limited to, or a follistatin polypeptide as well as variants thereof. In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 antibody antagonist (antibodies that inhibit one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and/or ALK7). In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 small molecule antagonist (e.g., small molecules that inhibit one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and/or ALK7). In some embodiments, an ActRII-ALK4 antagonist comprises an ActRII-ALK4 polynucleotide antagonist (e.g., nucleotide sequences that inhibit one or more of activin A, activin B, GDF8, GDF11, BMP6, BMP10, ActRIIB, ActRIIA, ALK4 and/or ALK7).

The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the 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.

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.

“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 the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % 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 authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 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 should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

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

“Antagonize”, in all its grammatical forms, refers to the process of inhibiting a protein and/or gene (e.g., by inhibiting or decreasing that protein's gene expression or by inducing an active protein to enter an inactive state) or decreasing a protein's and/or gene's activity.

The terms “about” and “approximately” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is ±10%. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably ≤5-fold and more preferably ≤2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers defining the ranges.

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”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein. Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two or more specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as 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).

Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.

2. ActRII-ALK4 Ligand Trap Antagonists and Variants Thereof

In certain aspects, an ActRII-ALK4 antagonist to be 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 (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure) is an ActRII-ALK4 ligand trap polypeptide including variants thereof as well as heterodimers and heteromultimers thereof. ActRII-ALK4 ligand trap polypeptides include TGF-β superfamily-related proteins, including variants thereof, that are capable of binding to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and BMP10). Therefore, ActRII-ALK4 ligand trap generally include polypeptides that are capable of antagonizing one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, and BMP10). For example, in some embodiments, an ActRII-ALK4 ligand trap comprises an ActRII polypeptide, including variants thereof, as well as homo- and hetero-multimers thereof (e.g., homodimer and heterodimers, respectively). As used herein, the term “ActRII” refers to the family of type II activin receptors. This family includes activin receptor type IIA (ActRIIA) and activin receptor type IIB (ActRIIB). In some embodiments, an ActRII-ALK4 ligand trap comprises an ActRIIB polypeptide, including variants thereof, as well has homomultimers (e.g., ActRIIB homodimers) and heteromultimers (e.g., ActRIIB-ALK4 or ActRIIB-ALK7 heterodimers). In some embodiments, an ActRII-ALK4 ligand trap comprises an ActRIIA polypeptide, including variants thereof, as well has homomultimers (e.g., ActRIIA homodimers) and heteromultimers (e.g., ActRIIA-ALK4 or ActRIIA-ALK7 heterodimers). In other embodiments, an ActRII-ALK ligand trap comprises a soluble ligand trap protein including, but not limited to, or a follistatin polypeptide as well as variants thereof.

A) ActRIIB Polypeptides

In certain aspects, the disclosure relates to ActRII-ALK4 antagonists comprising an ActRIIB polypeptide, which includes fragments, functional variants, and modified forms thereof as well as uses thereof (e.g., of treating, preventing, or reducing the progression rate and/or severity of heart failure (HF) or one or more complications of HF). As used herein, the term “ActRIIB” refers to a family of activin receptor type IIB (ActRIIB) proteins from any species and variant polypeptides derived from such ActRIIB proteins by mutagenesis or other modifications (including, e.g., mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIB polypeptides are provided throughout the present disclosure as well as 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 herein by reference in their entirety. Reference to ActRIIB herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIB family are generally all transmembrane polypeptides, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase specificity. The amino acid sequence of human ActRIIB precursor polypeptide is shown in FIG. 2 (SEQ ID NO: 2) and below. Preferably, ActRIIB polypeptides to be used in accordance with the methods of the disclosure are soluble. The term “soluble ActRIIB polypeptide,” as used herein, includes any naturally occurring extracellular domain of an ActRIIB polypeptide as well as any variants thereof (including mutants, fragments and peptidomimetic forms) that retain a 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 an ActRIIB extracellular domain (SEQ ID NO: 1) shown in FIG. 3 as well as SEQ ID NO: 53. This truncated ActRIIB extracellular domain (SEQ ID NO: 53) is denoted ActRIIB(25-131) based on numbering in SEQ ID NO: 2. Other examples of soluble ActRIIB polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRIIB polypeptide (see Example 4). The signal sequence can be a native signal sequence of an ActRIIB, or a signal sequence from another polypeptide, such as a tissue plasminogen activator (TPA) signal sequence or a honey bee melatin signal sequence. In some embodiments, ActRIIB polypeptides inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIB polypeptides bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Various examples of methods and assays for determining the ability for an ActRIIB polypeptide to bind to and/or inhibit activity of one or more ActRII-ALK4 ligands are disclosed herein or otherwise well known in the art, which can be readily used to determine if an ActRIIB polypeptide has the desired binding and/or antagonistic activities. Numbering of amino acids for all ActRIIB-related polypeptides described herein is based on the numbering of the human ActRIIB precursor protein sequence provided below (SEQ ID NO: 2), unless specifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 2, FIG. 2)
1   MTAPWVALAL LWGSLCAGSG RGEAETRECI YYNANWELER TQSGLERCE
51  GEQDKRLHCY ASWR                            SSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY
101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS
151 LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR
201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA
251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY
301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK
351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC
401 KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL
451 AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV
501 TNVDLPPKES SI

The signal peptide is indicated with a single underline; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated with a double underline.

A processed (mature) extracellular ActRIIB polypeptide sequence is as follows:

(SEQ ID NO: 1, FIG. 3)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSG
TIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP
EAGGPEVTYEPPPTAPT.

In some embodiments, the protein may be produced with an “SGR . . . ” sequence at the N-terminus. The C-terminal “tail” of the extracellular domain is indicated by a single underline. The sequence with the “tail” deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 386)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSG
TIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP
EA.

A form of ActRIIB with an alanine at position 64 of SEQ ID NO: 2 (A64) is also reported in the literature. See, e.g., Hilden et al. (1994) Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fc fusion protein comprising an extracellular domain of ActRIIB with the A64 substitution has a relatively low affinity for activin and GDF11. By contrast, the same ActRIIB-Fc fusion protein with an arginine at position 64 (R64) has an affinity for activin and GDF11 in the low nanomolar to high picomolar range. Therefore, sequences with an R64 are used as the “wild-type” reference sequence for human ActRIIB in this disclosure.

The form of ActRIIB with an alanine at position 64 is as follows:

(SEQ ID NO: 387)
1   MTAPWVALAL LWGSLCAGS              G RGEAETRECI YYNANWELER TNQSGLERCE
51  GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY
101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS
151 LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR
201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA
251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY
301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK
351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC
401 KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL
451 AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV
501 TNVDLPPKES SI

The signal peptide is indicated by single underline and the extracellular domain is indicated by bold font.

A processed (mature) extracellular ActRIIB polypeptide sequence of the alternative A64 form is as follows:

(SEQ ID NO: 388)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSG
TIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP
EAGGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ” sequence at the N-terminus. The C-terminal “tail” of the extracellular domain is indicated by single underline. The sequence with the “tail” deleted (a A15 sequence) is as follows:

(SEQ ID NO: 389)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSG
TIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP
EA

A nucleic acid sequence encoding the human ActRIIB precursor protein is shown below (SEQ ID NO: 4), representing nucleotides 25-1560 of GenBank Reference Sequence NM_001106.3, which encode amino acids 1-513 of the ActRIIB precursor. The sequence as shown provides an arginine at position 64 and may be modified to provide an alanine instead. The signal sequence is underlined.

(SEQ ID NO: 4, FIG. 4)
1    ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC
51   CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG
101  CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA
151  GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC
201  TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT
251  GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC
301  TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC
351  AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA
401  CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC
451  CTCATCGTCC TGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA
501  CGGTCATGTG GACATCCATG AGGACCCTGG GCCTCCACCA CCATCCCCTC
551  TGGTGGGCCT GAAGCCACTG CAGCTGCTGG AGATCAAGGC TCGGGGGCGC
601  TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTG TAGCTGTCAA
651  GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT
701  TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC
751  GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT
801  CCATGACAAG GGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT
851  GGAACGAACT GTGTCATGTA GCAGAGACGA TGTCACGAGG CCTCTCATAC
901  CTGCATGAGG ATGTGCCCTG GTGCCGTGGC GAGGGCCACA AGCCGTCTAT
951  TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAG AGCGACCTCA
1001 CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA
1051 CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC
1101 TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA
1151 TTGACATGTA TGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC
1201 AAGGCTGCAG ACGGACCCGT GGATGAGTAC ATGCTGCCCT TTGAGGAAGA
1251 GATTGGCCAG CACCCTTCGT TGGAGGAGCT GCAGGAGGTG GTGGTGCACA
1301 AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACA CCCGGGCCTG
1351 GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC
1401 TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT
1451 CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC
1501 ACCAATGTGG ACCTGCCCCC TAAAGAGTCA AGCATC

A nucleic acid sequence encoding a processed extracellular human ActRIIB polypeptide is as follows (SEQ ID NO: 3). The sequence as shown provides an arginine at position 64, and may be modified to provide an alanine instead (See FIG. 5, SEQ ID NO: 3).

(SEQ ID NO: 3)
1   GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG
51  GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC
101 AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC
151 ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA
201 TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT
251 GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTT GCCAGAGGCT
301 GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC

B) Variant ActRHB Polypeptides

In certain specific embodiments, the present disclosure contemplates making mutations in the extracellular domain (also referred to as ligand-binding domain) of an ActRIIB polypeptide such that the variant (or mutant) ActRIIB polypeptide has altered ligand-binding activities (e.g., binding affinity or binding selectivity). In certain cases, such variant ActRIIB polypeptides have altered (elevated or reduced) binding affinity for a specific ligand. In other cases, the variant ActRIIB polypeptides have altered binding selectivity for their ligands. For example, the disclosure provides a number of variant ActRIIB polypeptides that have reduced binding affinity to BMP9, compared to a non-modified ActRIIB polypeptide, but retain binding affinity for one or more of activin A, activin B, GDF8, GDF11, and BMP10. Optionally, the variant ActRIIB polypeptides have similar or the same biological activities of their corresponding wild-type ActRIIB polypeptides. For example, a variant ActRIIB polypeptide of the disclosure may bind to and inhibit function of an ActRIIB ligand (e.g., activin A, activin B, GDF8, GDF11 or BMP10). In some embodiments, a variant ActRIIB polypeptide of the disclosure treats, prevents, or reduces the progression rate 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 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., 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 with and/or fused to) with any of polypeptides disclosed herein.

ActRIIB is well-conserved across nearly all vertebrates, with large stretches of the extracellular domain conserved completely. See, e.g., FIG. 6. Many of the ligands that bind to ActRIIB are also highly conserved. Accordingly, comparisons of ActRIIB sequences from various vertebrate organisms provide insights into residues that may be altered. Therefore, an active, human ActRIIB variant may include one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIB, or may include a residue that is similar to that in the human or other vertebrate sequence.

The disclosure identifies functionally active portions and variants of ActRIIB. Applicant has previously ascertained 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 the position corresponding to amino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity for activin and GDF11. By contrast, the same Fc fusion polypeptide with an arginine at position 64 (R64) has an affinity for activin and GDF-11 in the low nanomolar to high picomolar range. Therefore, a sequence with an R64 (SEQ ID NO: 2) is used as the wild-type reference sequence for human ActRIIB in this disclosure, and the numbering for the variants described herein are based on the numbering in SEQ ID NO: 2. Additionally, one of skill in the art can make any of the ActRIIB variants described herein in the A64 background.

A processed extracellular ActRIIB polypeptide sequence is shown in SEQ ID NO: 1 (see, e.g., FIG. 3). In some embodiments, a processed ActRIIB polypeptide may be produced with an “SGR . . . ” sequence at the N-terminus. In some embodiments, a processed ActRIIB polypeptide may be produced with a “GRG . . . ” sequence at the N-terminus. For example, it is expected that some constructs, if expressed with a TPA leader, will lack the N-terminal serine. Accordingly, mature ActRIIB sequences described herein may begin with either an N-terminal serine or an N-terminal glycine (lacking the N-terminal serine).

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that a deletion of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduced the affinity of the receptor for activin. Data disclosed in WO2008097541 show that an ActRIIB-Fc fusion polypeptide containing amino acids 20-119 of SEQ ID NO: 2, “ActRIIB(20-119)-Fc” has reduced binding to GDF11 and activin relative to an ActRIIB(20-134)-Fc, which includes the proline knot region and the complete juxtamembrane domain. However, an ActRIIB(20-129)-Fc polypeptide retains similar but somewhat reduced activity relative to the wild type, even though the proline knot region is disrupted. Thus, ActRIIB extracellular domains that stop at amino acid 134, 133, 132, 131, 130 and 129 are all expected to be active, but constructs stopping at 134 or 133 may be most active. Similarly, mutations at any of residues 129-134 are not expected to alter ligand binding affinity by large margins. In support of this, mutations of P129 and P130 do not substantially decrease ligand binding. Therefore, an ActRIIB-Fc fusion polypeptide may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 are expected to have reduced ligand binding. Amino acid 119 is poorly conserved and so is readily altered or truncated. Forms ending at 128 or later retain ligand binding activity. Forms ending at or between 119 and 127 will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a polypeptide beginning at amino acid 29 or before will retain ligand binding activity. Amino acid 29 represents the initial cysteine. An alanine-to-asparagine mutation 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 cross-linked region, corresponding to amino acids 20-29, are well tolerated. In particular, constructs beginning at position 20, 21, 22, 23 and 24 will retain activity, and constructs beginning at positions 25, 26, 27, 28 and 29 are also expected to retain activity. Data shown in WO2008097541 demonstrate that, surprisingly, a construct beginning at 22, 23, 24 or 25 will have the most activity.

Taken together, an active portion of ActRIIB comprises amino acids 29-109 of SEQ ID NO: 2, and constructs may, for example, begin at a residue corresponding to amino acids 20-29 and end at a position corresponding to amino acids 109-134. Other examples include constructs that begin at a position from 20-29 or 21-29 and end at a position from 119-134, 119-133 or 129-134, 129-133. Other examples include constructs that begin at a position from 20-24 (or 21-24, or 22-25) and end at a position from 109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or 129-133). Variants within these ranges are also contemplated, particularly those 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.

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

It has been previously demonstrated that the addition of a further N-linked glycosylation site (N-X-S/T) into the ActRIIB extracellular domain is well-tolerated (see, e.g., U.S. Pat. No. 7,842,663). Therefore, N-X-S/T sequences may be generally introduced at positions outside the ligand binding pocket defined in FIG. 1 in ActRIIB polypeptide of the present disclosure. Particularly suitable sites for the introduction of non-endogenous N-X-S/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). N-X-S/T sequences may also be introduced into the linker between the ActRIIB sequence and an Fc domain or other fusion component as well as optionally into the fusion component itself. Such a site may be introduced with minimal effort by introducing an N in the correct position with respect to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N. Thus, desirable alterations that would create an N-linked glycosylation site are: A24N, R64N, S67N (possibly combined with an N65A alteration), E105N, RI 12N, G120N, E123N, P129N, A132N, R112S and R112T (with respect to SEQ ID NO: 2). Any S that is predicted to be glycosylated may be altered to a T without creating an immunogenic site, because of the protection afforded by the glycosylation. Likewise, any T that is predicted to be glycosylated may be altered to an S. Thus, the alterations S67T and S44T (with respect to SEQ ID NO: 2) are contemplated. Likewise, in an A24N variant, an S26T alteration may be used. Accordingly, an ActRIIB polypeptide of the present disclosure may be a variant having one or more additional, non-endogenous N-linked glycosylation consensus sequences as described above.

In certain embodiments, a variant ActRIIB polypeptide has an amino acid sequence that is 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.

In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 any one 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, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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. An ActRIIB-Fc fusion protein comprising SEQ ID NO: 31 may optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%0, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%0, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%0, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%0, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist 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 optionally be provided with the lysine removed from the C-terminus. In some embodiments, variant ActRIIB polypeptides or variant ActRIIB-Fc fusion polypeptides of the disclosure comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%⁰, 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 optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the disclosure relates to variant ActRIIB polypeptides 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 that begins at any one of amino acids 20-29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acids 109-134 (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 wherein the polypeptide comprises one or more amino acid substitutions at a position of SEQ ID NO: 2 selected from the group consisting of: K55, F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 as well as heteromultimer complexes comprising one or more such variant ActRIIB polypeptides. In certain aspects, the disclosure relates to variant ActRIIB polypeptides 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 that begins at any one of amino acids 20-29 (e.g., amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 2 and ends at any one of amino acids 109-134 (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 wherein the polypeptide comprises one or more amino acid substitutions at a position of SEQ ID NO: 2, but wherein the amino acid at position corresponding to 79 of SEQ ID NO:2 is leucine as well as heteromultimer complexes comprising one or more such variant ActRIIB polypeptides. In some embodiments, the variant 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, the variant 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, the variant 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, the variant 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, the variant 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: 12. In some embodiments, the variant 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: 5. In some embodiments, the polypeptide comprises an amino acid substitution at the 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 the 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 the 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 the amino acid position corresponding to E37 of 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 the amino acid position corresponding to L38 of SEQ ID NO: 2. For example, in some embodiments, the substitution is L38N. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to R40 of 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 the amino acid position corresponding to S44 of SEQ ID NO: 2. For example, in some embodiments, the substitution is S44T. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to L46 of 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 the amino acid position corresponding to E50 of 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 the 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 the 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 the 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 the 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 the 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 the 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 the amino acid position corresponding to Y60 of 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 the amino acid position corresponding to R64 of 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 the amino acid position corresponding to N65 of SEQ ID NO: 2. For example, in some embodiments, the substitution is N65A. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to S67 of 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 the amino acid position corresponding to G68 of SEQ ID NO: 2. For example, in some embodiments, the substitution is G68R. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to K74 of 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 the amino acid position corresponding to W78 of 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 the amino acid position corresponding to L79 of SEQ ID NO: 2. For example, in some embodiments, the substitution is L79D. In some embodiments, the substitution does not comprise an acidic amino acid at the 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 comprise an aspartic acid (D) at the 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 the 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 of 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 the amino acid position corresponding to N83 of 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 the amino acid position corresponding to T93 of 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 the amino acid position corresponding to E94 of SEQ ID NO: 2. For example, in some embodiments, the substitution is E94K. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to Q98 of 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 the amino acid position corresponding to V99 of 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 the amino acid position corresponding to E105 of SEQ ID NO: 2. For example, in some embodiments, the substitution is E105N. In some embodiments, the polypeptide comprises an amino acid substitution at the 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 the 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 the 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 the 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 the amino acid position corresponding to A119 of 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 the amino acid position corresponding to G120 of SEQ ID NO: 2. For example, in some embodiments, the substitution is G120N. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to E123 of SEQ ID NO: 2. For example, in some embodiments, the substitution is E123N. In some embodiments, the polypeptide comprises an amino acid substitution at the amino acid position corresponding to P129 of 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.

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, the variant ActRIIB polypeptide comprises a substitution at position A24 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position E37 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position R40 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position D54 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position K55 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position R56 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position R64 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position K74 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position W78 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position L79 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position D80 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position F82 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position P129 with respect to SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a substitution at position P130 with respect to SEQ ID NO: 2.

In certain aspects, the 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 the position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 31 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 33. In some embodiments, the variant ActRIIB polypeptide comprises an alanine at the position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 33 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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 the position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 34 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 36. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to K55 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 36 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 37. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 37 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 39. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 39 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 40. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 40 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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 the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 42 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 43. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 43 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 45. In some embodiments, the variant ActRIIB polypeptide comprises a glutamic acid at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 45 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 336. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 336 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 338. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 338 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 342. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 342 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 344. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 344 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 348. In some embodiments, the variant ActRIIB polypeptide comprises a leucine at the position corresponding to E50 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 348 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 350. In some embodiments, the variant ActRIIB polypeptide comprises a leucine at the position corresponding to E50 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 350 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 354. In some embodiments, the variant ActRIIB polypeptide comprises a glycine at the position corresponding to V99 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 354 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 356. In some embodiments, the variant ActRIIB polypeptide comprises a glycine at the position corresponding to V99 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 356 may optionally be provided with the lysine removed from the C-terminus.

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 2 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 3 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 4 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 5 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 6 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 7 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 8 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 9 of any of the amino acid substitutions disclosed herein. In some embodiments, any of the variant ActRIIB polypeptides disclosed herein comprises 10 of any of the amino acid substitutions disclosed herein.

In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising two or more amino acid substitutions as compared to the reference amino acid sequence of SEQ ID NO: 2. For example, in some embodiments, the variant ActRIIB polypeptide comprises an A24N substitution and a K74A substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L79P substitution and a K74A substitution. In some embodiments, the variant ActRIIB polypeptide comprises a P129S substitution and a PT30A substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L38N substitution and a L79R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82I substitution and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82K substitution and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82T substitution and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L79H substitution and a F82K substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L79H substitution and a F82I substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82D substitution and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82E substitution and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L79F substitution and a F82D substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L79F substitution and a F82T substitution. In some embodiments, the variant ActRIIB polypeptide comprises a E52D substitution and a F82D substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52D substitution and a F82T substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L57R substitution and a F82D substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L57R substitution and a F82T substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82I substitution and an E94K substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82S substitution and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L57R substitution and a F82S substitution. In some embodiments, the variant ActRIIB polypeptide comprises a K74A substitution and a L79P substitution. In some embodiments, the variant ActRIIB polypeptide comprises a K55A substitution and a F82I substitution. In some embodiments, the variant ActRIIB polypeptide comprises a L79K substitution and a F82K substitution. In some embodiments, the variant ActRIIB polypeptide comprises a F82W substitution and a N83A substitution.

In certain aspects, the 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: 276. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2 and an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 276 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 278. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2 and an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 278 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 279. In some embodiments, the variant ActRIIB polypeptide comprises an lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at the position corresponding to F82 of SEQ ID NO: 2 and an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 279 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 332. In some embodiments, the variant ActRIIB polypeptide comprises an lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at the position corresponding to F82 of SEQ ID NO: 2 and an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 332 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 333. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at the position corresponding to F82 of SEQ ID NO: 2 and an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 333 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 335. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a threonine at the position corresponding to F82 of SEQ ID NO: 2 and an arginine at the position corresponding to N83 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 335 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 339. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2 and an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 339 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 341. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2 and an isoleucine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 341 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 345. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2, and a lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 345 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 347. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises a histidine at the position corresponding to L79 of SEQ ID NO: 2, and a lysine at the position corresponding to F82 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 347 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 351. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at the position corresponding to L38 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at the position corresponding to L38 of SEQ ID NO: 2, and an arginine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 351 may optionally be provided with the lysine removed from the C-terminus.

In certain aspects, the 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: 353. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at the position corresponding to L38 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an arginine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the variant ActRIIB polypeptide comprises an asparagine at the position corresponding to L38 of SEQ ID NO: 2, and an arginine at the position corresponding to L79 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of SEQ ID NO: 353 may optionally be provided with the lysine removed from the C-terminus.

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

In certain aspects, the disclosure relates to a variant ActRIIB polypeptide comprising four or more amino acid substitutions as 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, a L79E substitution, a F82Y substitution, and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, a L79E substitution, a F82T substitution, and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises a G68R substitution, a L79T substitution, a F82T substitution, and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide comprises an E52N substitution, a G68R substitution, a F82Y substitution, and a N83R substitution. In some embodiments, the variant ActRIIB polypeptide may comprise at least two of the amino acid substitutions described in any of the variant ActRIIB polypeptides above. In some embodiments, the variant ActRIIB polypeptide may comprise at least three of the amino acid substitutions described in any of the variant ActRIIB polypeptides above.

C) ActRIIA Polypeptides

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

The canonical human ActRIIA precursor protein sequence is as follows:

(SEQ ID NO: 366)
1   MGAAAKLAFA VFLISCSSGA ILGRSETQEC LFFNANWEKD RTQTGVEPC
51  YGDKDKRRHC FATWK                            ISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV
101 YFCCCEGNMC NEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI
151 AGIVICAFWV YRHHKMAYPP VLVPTQDPGP PPPSPLLGLK PLQLLEVKAR
201 GREGCVWKAQ LLNEYVAVKI FPIQDKQSWQ NEYEVYSLPG MKHENILQFI
251 GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANVVSWNELC HIAETMARGL
301 AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FGLALKFEAG
351 KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR
401 CTAADGPVDE YMLPFEEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG
451 MAMLCETIEE CWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM
501 VTNVDFPPKE SSL

The signal peptide is indicated by a single underline; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated by a double underline.

A processed (mature) extracellular human ActRIIA polypeptide sequence is as follows:

(SEQ ID NO: 367)
ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISG
SIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP
EMEVTQPTSNPVTPKPP

The C-terminal “tail” of the extracellular domain is indicated by single underline. The sequence with the “tail” deleted (a A15 sequence) is as follows:

(SEQ ID NO: 368)
ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISG
SIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP
EM

A nucleic acid sequence encoding human ActRIIA precursor protein is shown below (SEQ ID NO: 369), as follows nucleotides 159-1700 of GenBank Reference Sequence NM_001616.4. The signal sequence is underlined.

(SEQ ID NO: 369)
1    ATGGGAGCTG CTGCAAAGTT GGCGTTTGCC GTCTTTCTTA TCTCCTGTTC
51   TTCAGGTGCT ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA
101  ATGCTAATTG GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT
151  TATGGTGACA AAGATAAACG GCGGCATTGT TTTGCTACCT GGAAGAATAT
201  TTCTGGTTCC ATTGAAATAG TGAAACAAGG TTGTTGGCTG GATGATATCA
251  ACTGCTATGA CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA
301  TATTTTTGTT GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT
351  TCCGGAGATG GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC
401  CACCCTATTA CAACATCCTG CTCTATTCCT TGGTGCCACT TATGTTAATT
451  GCGGGGATTG TCATTTGTGC ATTTTGGGTG TACAGGCATC ACAAGATGGC
501  CTACCCTCCT GTACTTGTTC CAACTCAAGA CCCAGGACCA CCCCCACCTT
551  CTCCATTACT AGGTTTGAAA CCACTGCAGT TATTAGAAGT GAAAGCAAGG
601  GGAAGATTTG GTTGTGTCTG GAAAGCCCAG TTGCTTAACG AATATGTGGC
651  TGTCAAAATA TTTCCAATAC AGGACAAACA GTCATGGCAA AATGAATACG
701  AAGTCTACAG TTTGCCTGGA ATGAAGCATG AGAACATATT ACAGTTCATT
751  GGTGCAGAAA AACGAGGCAC CAGTGTTGAT GTGGATCTTT GGCTGATCAC
801  AGCATTTCAT GAAAAGGGTT CACTATCAGA CTTTCTTAAG GCTAATGTGG
851  TCTCTTGGAA TGAACTGTGT CATATTGCAG AAACCATGGC TAGAGGATTG
901  GCATATTTAC ATGAGGATAT ACCTGGCCTA AAAGATGGCC ACAAACCTGC
951  CATATCTCAC AGGGACATCA AAAGTAAAAA TGTGCTGTTG AAAAACAACC
1001 TGACAGCTTG CATTGCTGAC TTTGGGTTGG CCTTAAAATT TGAGGCTGGC
1051 AAGTCTGCAG GCGATACCCA TGGACAGGTT GGTACCCGGA GGTACATGGC
1101 TCCAGAGGTA TTAGAGGGTG CTATAAACTT CCAAAGGGAT GCATTTTTGA
1151 GGATAGATAT GTATGCCATG GGATTAGTCC TATGGGAACT GGCTTCTCGC
1201 TGTACTGCTG CAGATGGACC TGTAGATGAA TACATGTTGC CATTTGAGGA
1251 GGAAATTGGC CAGCATCCAT CTCTTGAAGA CATGCAGGAA GTTGTTGTGC
1301 ATAAAAAAAA GAGGCCTGTT TTAAGAGATT ATTGGCAGAA ACATGCTGGA
1351 ATGGCAATGC TCTGTGAAAC CATTGAAGAA TGTTGGGATC ACGACGCAGA
1401 AGCCAGGTTA TCAGCTGGAT GTGTAGGTGA AAGAATTACC CAGATGCAGA
1451 GACTAACAAA TATTATTACC ACAGAGGACA TTGTAACAGT GGTCACAATG
1501 GTGACAAATG TTGACTTTCC TCCCAAAGAA TCTAGTCTA

A nucleic acid sequence encoding processed soluble (extracellular) human ActRIIA polypeptide is as follows:

(SEQ ID NO: 370)
1   ATACTTGGTA GATCAGAAAC TCAGGAGTGT CTTTTCTTTA
    ATGCTAATTG
51  GGAAAAAGAC AGAACCAATC AAACTGGTGT TGAACCGTGT
    TATGGTGACA
101 AAGATAAACG GCGGCATTGT TTTGCTACCT GGAAGAATAT
    TTCTGGTTCC
151 ATTGAAATAG TGAAACAAGG TTGTTGGCTG GATGATATCA
    ACTGCTATGA
201 CAGGACTGAT TGTGTAGAAA AAAAAGACAG CCCTGAAGTA
    TATTTTTGTT
251 GCTGTGAGGG CAATATGTGT AATGAAAAGT TTTCTTATTT
    TCCGGAGATG
301 GAAGTCACAC AGCCCACTTC AAATCCAGTT ACACCTAAGC
    CACCC

ActRIIA is well-conserved among vertebrates, with large stretches of the extracellular domain completely conserved. For example, FIG. 10 depicts a multi-sequence alignment of a human ActRIIA extracellular domain (SEQ ID NO: 367) compared to various ActRIIA orthologs (SEQ ID NOs: 371-377). Many of the ligands that bind to ActRIIA are also highly conserved. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIA-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ActRIIA-ligand binding activities. Therefore, an active, human ActRIIA variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIA, or may include a residue that is similar to that in the human or other vertebrate sequences.

Without meaning to be limiting, the following examples illustrate this approach to defining an active ActRIIA variant. As illustrated in FIG. 10, F13 in 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 are tolerated at this position, including F, W, and Y. Q24 in the human extracellular domain is R in Bos Taurus ActRIIA, indicating that charged residues will be tolerated at this position, including D, R, K, H, and E. S95 in the human extracellular domain is F in Gallus gallus and Tyto alba ActRIIA, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y, and probably hydrophobic residue such as L, I, or F. E52 in the human extracellular domain is D in Ovis aries ActRIIA, indicating that acidic residues are tolerated at this position, including D and E. P29 in the human extracellular domain is relatively poorly conserved, appearing as S in Ovis aries ActRIIA and L in Myotis davidii ActRIIA, thus essentially any amino acid should be tolerated at this position.

Moreover, as discussed above, ActRII proteins have been characterized in the art in terms of structural/functional characteristics, particularly with respect to ligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; as well as U.S. Pat. Nos. 7,709,605, 7,612,041, and 7,842,663]. In addition to the teachings herein, these references provide amply guidance for how to generate ActRII variants that retain one or more desired activities (e.g., ligand-binding activity).

For example, a defining structural motif known as a three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine residues located at varying 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]. Accordingly, the core ligand-binding domains of human ActRIIA, as demarcated by the outermost of these conserved cysteines, corresponds to positions 30-110 of SEQ ID NO: 366 (ActRIIA precursor). Therefore, the structurally less-ordered amino acids flanking these cysteine-demarcated 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 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, or 25 residues at the C-terminus without necessarily altering ligand binding. Exemplary ActRIIA extracellular domains truncations include SEQ ID NOs: 367 and 368.

Accordingly, a general formula for an active portion (e.g., ligand binding) of ActRIIA is a polypeptide that comprises, consists essentially of, or consists of amino acids 30-110 of SEQ ID NO: 366. Therefore ActRIIA polypeptides may, for example, comprise, consists essentially of, or consists 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 a portion of ActRIIA beginning at a residue corresponding to any one of amino acids 21-30 (e.g., beginning at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of SEQ ID NO: 366 and ending at a position corresponding to any one amino acids 110-135 (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, 131, 132, 133, 134, or 135) of SEQ ID NO: 366. Other examples include constructs that begin at a position selected from 21-30 (e.g., beginning at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), 22-30 (e.g., beginning at any one of amino acids 22, 23, 24, 25, 26, 27, 28, 29, or 30), 23-30 (e.g., beginning at any one of amino acids 23, 24, 25, 26, 27, 28, 29, or 30), 24-30 (e.g., beginning at any one of amino acids 24, 25, 26, 27, 28, 29, or 30) of SEQ ID NO: 366, and end at a position selected from 111-135 (e.g., ending at 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), 112-135 (e.g., ending at 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), 113-135 (e.g., ending at 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), 120-135 (e.g., ending at any one of amino acids 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135), 130-135 (e.g., ending at any one of amino acids 130, 131, 132, 133, 134 or 135), 111-134 (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, 131, 132, 133, or 134), 111-133 (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, 131, 132, or 133), 111-132 (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, 131, or 132), or 111-131 (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, or 131) of SEQ ID NO: 366. Variants within these ranges are also contemplated, particularly those comprising, consisting essentially of, or consisting of an amino acid sequence that has 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, an ActRIIA polypeptide may comprise, consists 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. Optionally, ActRIIA polypeptides comprise 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, and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand-binding pocket. In some embodiments, ActRIIA polypeptide of the disclosure comprise, consist essentially of, or consist 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 a portion of ActRIIA beginning at a residue corresponding to amino acids 21-30 (e.g., beginning at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of SEQ ID NO: 366 and ending at a position corresponding to any one amino acids 110-135 (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, 131, 132, 133, 134 or 135) of SEQ ID NO: 366. In some embodiments, ActRIIA polypeptides comprise, consist, or consist 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 amino acids 30-110 of SEQ ID NO: 366. In certain embodiments, ActRIIA polypeptides comprise, consist, or consist 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 amino acids 21-135 of SEQ ID NO: 366. In some embodiments, ActRIIA polypeptides comprise, consist, or consist 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, ActRIIA polypeptides comprise, consist, or consist 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, ActRIIA polypeptides comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%8, 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, ActRIIA polypeptides comprise, consist, or consist 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, ActRIIA polypeptides comprise, consist, or consist 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, ActRIIA polypeptides comprise, consist, or consist 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: 384. In some embodiments, ActRIIA polypeptides comprise, consist, or consist essentially of an amino acid sequence that is at least 70%, 75%, 80%, 85%8, 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, ActRIIA polypeptides comprise, consist, or consist 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.

D) ALK4 Polypeptides

In certain aspects, the disclosure relates to ActRII-ALK4 antagonists comprising an ALK4 polypeptide, which includes fragments, functional variants, and modified forms thereof as well as uses thereof (e.g., of treating, preventing, or reducing the progression rate and/or severity of heart failure (HF) or one or more complications of HF). As used herein, the term “ALK4” refers to a family of activin receptor-like kinase-4 (ALK4) proteins from any species and variant polypeptides derived from such ALK4 proteins by mutagenesis or other modifications (including, e.g., mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ALK4 polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication Nos. WO/2016/164089, WO/2016/164497, and WO/2018/067879, which are incorporated herein by reference in their entirety. Reference to ALK4 herein is understood to be a reference 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, ALK4 polypeptides to be used in accordance with the methods of the disclosure are soluble. The term “soluble ALK4 polypeptide,” as used herein, includes any naturally occurring extracellular domain of an ALK4 polypeptide as well as any variants thereof (including mutants, fragments and peptidomimetic forms) that retain a useful activity. For example, the extracellular domain of an ALK4 polypeptide binds to a ligand and is generally soluble. Examples of soluble ALK4 polypeptides include an ALK4 extracellular domain (SEQ ID NO: 86) shown below, Other examples of soluble ALK4 polypeptides comprise a signal sequence in addition to the extracellular domain of an ALK4 polypeptide. The signal sequence can be a native signal sequence of an ALK4, or a signal sequence from another polypeptide, such as a tissue plasminogen activator (TPA) signal sequence or a honey bee melatin signal sequence. In some embodiments, ALK4 polypeptides inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ALK4 polypeptides bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Various examples of methods and assays for determining the ability for an ALK4 polypeptide to bind to and/or inhibit activity of one or more ActRII-ALK4 ligands are disclosed herein or otherwise well known in the art, which can be readily used to determine if an ActRIIB polypeptide has the desired binding and/or antagonistic activities. Numbering of amino acids for all ALK4-related polypeptides described herein is based on the numbering of the human ALK4 precursor protein sequence provided below (SEQ ID NO: 84), unless specifically designated otherwise.

A human ALK4 precursor polypeptide sequence (NCBI Ref Seq NP 004293) is as follows:

(SEQ ID NO: 84)
1   MAESAGASSF FPLVVLLLAG SGG              SGPRGVQ ALLCACTSCL
    QANYTCETDG ACMVSIFNLD
61  GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDY
    CNRIDLRVPS GHLKEPEHPS
121 MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ
    RLDMEDPSCE MCLSKDKTLQ
181 DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE
    VWRGRWRGGD VAVKIFSSRE
241 ERSWFREAEI YQTVMLRHEN ILGFIAADNK DNGTWTQLWL
    VSDYHEHGSL FDYLNRYTVT
301 IEGMIKLALS AASGLAHLHM EIVGTQGKPG IAHRDLKSKN
    ILVKKNGMCA IADLGLAVRH
361 DAVIDTIDIA PNQRVGTKRY MAPEVLDETI NMKHFDSFKC
    ADIYALGLVY WEIARRCNSG
421 GVHEEYQLPY YDLVPSDPSI EEMRKVVCDQ KLRPNIPNWW
    QSYEALRVMG KMMRECWYAN
481 GAARLTALRI KKTLSQLSVQ EDVKI

The signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.

A processed extracellular human ALK4 polypeptide sequence is as follows:

(SEQ ID NO: 86)
SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKV
ELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWG
PVE

A nucleic acid sequence encoding an ALK4 precursor polypeptide is shown in SEQ ID NO: 221), corresponding to nucleotides 78-1592 of GenBank Reference Sequence NM_004302.4.

The signal sequence is underlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 221)
ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCT
GCTCGCCGGCAGCGGCGGG              TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGT
GTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGG
GCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCG
CACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACT
GCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTAC
TGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGA
GCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCC
CGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAAC
TATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCC
CTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCT
ACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAG
CGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCG
GTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGA
AAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATA
TACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGC
TGACAATAAAGATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACT
ATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACA
ATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACA
CCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATC
GAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCC
ATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCAT
TGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTG
AAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGT
GCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATG
CAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAG
TGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAG
AAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCG
GGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCC
GCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAG
GAAGACGTGAAGATC

A nucleic acid sequence encoding the extracellular ALK4 polypeptide is shown in SEQ ID NO: 222.

(SEQ ID NO: 222)
TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCT
CCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTT
TCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTG
GAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCT
GCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGA
GGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC
CCGGTGGAG

An alternative isoform of human ALK4 precursor protein sequence, isoform B (NCBI Ref Seq NP_064732.3), is as follows:

(SEQ ID NO: 421)
1   MVSIFNLDGM EHHVRTCIPK VELVPAGKPF YCLSSEDLRN
    THCCYTDYCN RIDLRVPSGH
61  LKEPEHPSMW GPVELVGIIA GPVFLLFLII IIVFLVINYH
    QRVYHNRQRL DMEDPSCEMC
121 LSKDKTLQDL VYDLSTSGSG SGLPLFVQRT VARTIVLQEI
    IGKGRFGEVW RGRWRGGDVA
181 VKIFSSREER SWFREAEIYQ TVMLRHENIL GFIAADNKDN
    GTWTQLWLVS DYHEHGSLFD
241 YLNRYTVTIE GMIKLALSAA SGLAHLHMEI VGTQGKPGIA
    HRDLKSKNIL VKKNGMCAIA
301 DLGLAVRHDA VTDTIDIAPN QRVGTKRYMA PEVLDETINM
    KHFDSFKCAD IYALGLVYWE
361 IARRCNSGGV HEEYQLPYYD LVPSDPSIEE MRKVVCDQKL
    RPNIPNWWQS YEALRVMGKM
421 MRECWYANGA ARLTALRIKK TLSQLSVQED VKI

The extracellular domain is indicated in bold font.

A processed extracellular ALK4 polypeptide sequence is as follows:

• • 1 MVSIFNLDGM EHHVRTCIPK VELVPAGKPF YCLSSEDLRN THCCYTDYCN RIDLRVPSGH 61 LKEPEHPSMW GPVE (SEQ ID NO: 422)

A 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. The nucleotides encoding the extracellular domain are indicated in bold font.

(SEQ ID NO: 423)
1    ATGGTTTCCA TTTTCAATCT GGATGGGATG GAGCACCATG
     TGCGCACCTG
51   CATCCCCAAA GTGGAGCTGG TCCCTGCCGG GAAGCCCTTC
     TACTGCCTGA
101  GCTCGGAGGA CCTGCGCAAC ACCCACTGCT GCTACACTGA
     CTACTGCAAC
151  AGGATCGACT TGAGGGTGCC CAGTGGTCAC CTCAAGGAGC
     CTGAGCACCC
201  GTCCATGTGG GGCCCGGTGG AGCTGGTAGG CATCATCGCC
     GGCCCGGTGT
251  TCCTCCTGTT CCTCATCATC ATCATTGTTT TCCTTGTCAT
     TAACTATCAT
301  CAGCGTGTCT ATCACAACCG CCAGAGACTG GACATGGAAG
     ATCCCTCATG
351  TGAGATGTGT CTCTCCAAAG ACAAGACGCT CCAGGATCTT
     GTCTACGATC
401  TCTCCACCTC AGGGTCTGGC TCAGGGTTAC CCCTCTTTGT
     CCAGCGCACA
451  GTGGCCCGAA CCATCGTTTT ACAAGAGATT ATTGGCAAGG
     GTCGGTTTGG
501  GGAAGTATGG CGGGGCCGCT GGAGGGGTGG TGATGTGGCT
     GTGAAAATAT
551  TCTCTTCTCG TGAAGAACGG TCTTGGTTCA GGGAAGCAGA
     GATATACCAG
601  ACGGTCATGC TGCGCCATGA AAACATCCTT GGATTTATTG
     CTGCTGACAA
651  TAAAGATAAT GGCACCTGGA CACAGCTGTG GCTTGTTTCT
     GACTATCATG
701  AGCACGGGTC CCTGTTTGAT TATCTGAACC GGTACACAGT
     GACAATTGAG
751  GGGATGATTA AGCTGGCCTT GTCTGCTGCT AGTGGGCTGG
     CACACCTGCA
801  CATGGAGATC GTGGGCACCC AAGGGAAGCC TGGAATTGCT
     CATCGAGACT
851  TAAAGTCAAA GAACATTCTG GTGAAGAAAA ATGGCATGTG
     TGCCATAGCA
901  GACCTGGGCC TGGCTGTCCG TCATGATGCA GTCACTGACA
     CCATTGACAT
951  TGCCCCGAAT CAGAGGGTGG GGACCAAACG ATACATGGCC
     CCTGAAGTAC
1001 TTGATGAAAC CATTAATATG AAACACTTTG ACTCCTTTAA
     ATGTGCTGAT
1051 ATTTATGCCC TCGGGCTTGT ATATTGGGAG ATTGCTCGAA
     GATGCAATTC
1101 TGGAGGAGTC CATGAAGAAT ATCAGCTGCC ATATTACGAC
     TTAGTGCCCT
1151 CTGACCCTTC CATTGAGGAA ATGCGAAAGG TTGTATGTGA
     TCAGAAGCTG
1201 CGTCCCAACA TCCCCAACTG GTGGCAGAGT TATGAGGCAC
     TGCGGGTGAT
1251 GGGGAAGATG ATGCGAGAGT GTTGGTATGC CAACGGCGCA
     GCCCGCCTGA
1301 CGGCCCTGCG CATCAAGAAG ACCCTCTCCC AGCTCAGCGT
     GCAGGAAGAC
1351 GTGAAGATCT AA

A nucleic acid sequence encoding the extracellular ALK4 polypeptide (isoform B) is as follows:

(SEQ ID NO: 424)
1   ATGGTTTCCA TTTTCAATCT GGATGGGATG GAGCACCATG
    TGCGCACCTG
51  CATCCCCAAA GTGGAGCTGG TCCCTGCCGG GAAGCCCTTC
    TACTGCCTGA
101 GCTCGGAGGA CCTGCGCAAC ACCCACTGCT GCTACACTGA
    CTACTGCAAC
151 AGGATCGACT TGAGGGTGCC CAGTGGTCAC CTCAAGGAGC
    CTGAGCACCC
201 GTCCATGTGG GGCCCGGTGG AGCTGGTAGG

An alternative isoform of human ALK4 precursor polypeptide sequence, isoform C (NCBI Ref Seq NP_064733.3), is as follows:

(SEQ ID NO: 85)
1   MAESAGASSF FPLVVLLLAG SGG              SGPRGVQ ALLCACTSCL
    QANYTCETDG ACMVSIFNLD
61  GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDY
    CNRIDLRVPS GHLKEPEHPS
121 MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ
    RLDMEDPSCE MCLSKDKTLQ
181 DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE
    VWRGRWRGGD VAVKIFSSRE
241 ERSWFREAEI YQTVMLRHEN ILGFIAADNK ADCSFLTLPW
    EVVMVSAAPK LRSLRLQYKG
301 GRGRARFLFP LNNGTWTQLW LVSDYHEHGS LFDYLNRYTV
    TIEGMIKLAL SAASGLAHLH
361 MEIVGTQGKP GIAHRDLKSK NILVKKNGMC AIADLGLAVR
    HDAVTDTIDI APNQRVGTKR
421 YMAPEVLDET INMKHFDSFK CADIYALGLV YWEIARRCNS
    GGVHEEYQLP YYDLVPSDPS
481 IEEMRKVVCD QKLRPNIPNW WQSYEALRVM GKMMRECWYA
    NGAARLTALR IKKTLSQLSV
541 QEDVKI

The signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.

A processed extracellular ALK4 polypeptide sequence (isoform C) is as follows:

(SEQ ID NO: 87)
SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKV
ELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWG
PVE

A nucleic acid sequence encoding an ALK4 precursor polypeptide (isoform C) is shown in SEQ ID NO: 223, corresponding to nucleotides 78-1715 of GenBank Reference Sequence NM_020328.3. A nucleic acid sequence encoding the extracellular ALK4 polypeptide (isoform C) is shown in SEQ ID NO: 224.

(SEQ ID NO: 223)
ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCT
GCTCGCCGGCAGCGGCGGG              TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGT
GTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGG
GCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCG
CACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACT
GCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTAC
TGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGA
GCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCC
CGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAAC
TATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCC
CTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCT
ACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAG
CGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCG
GTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGA
AAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATA
TACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGC
TGACAATAAAGCAGACTGCTCATTCCTCACATTGCCATGGGAAGTTGTAA
TGGTCTCTGCTGCCCCCAAGCTGAGGAGCCTTAGACTCCAATACAAGGGA
GGAAGGGGAAGAGCAAGATTTTTATTCCCACTGAATAATGGCACCTGGAC
ACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATT
ATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTG
TCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCA
AGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGG
TGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGT
CATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGG
GACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGA
AACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTA
TATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATA
TCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAA
TGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGG
TGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTG
TTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGA
CCCTCTCCCAGCTCAGCGTGCAGGAAGACGTGAAGATC
(SEQ ID NO: 224)
TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCT
CCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTT
TCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTG
GAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCT
GCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGA
GGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC
CCGGTGGAG

ALK4 is well-conserved among vertebrates, with large stretches of the extracellular domain completely conserved. For example, FIG. 9 depicts a multi-sequence alignment of a human ALK4 extracellular domain compared to various ALK4 orthologs. Many of the ligands that bind to ALK4 are also highly conserved. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ALK4-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ALK4-ligand binding activities. Therefore, an active, human ALK4 variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ALK4, or may include a residue that is similar to that in the human or other vertebrate sequences. Without meaning to be limiting, the following examples illustrate this approach to defining an active ALK4 variant. As illustrated in FIG. 9, V6 in the human ALK4 extracellular domain (SEQ ID NO: 414) is isoleucine in Mus muculus ALK4 (SEQ ID NO: 418), and so the position may be altered, and optionally may be altered to another hydrophobic residue such as L, I, or F, or a non-polar residue such as A, as is 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 be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y, and probably a non-polar residue such as A. S15 in the human extracellular domain is D in Gallus gallus ALK4, indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, T, R, E, K, H, G, P, G and Y. E40 in the human extracellular domain is K in Gallus gallus ALK4, indicating that charged residues will be tolerated at this position, including D, R, K, H, as well as Q and N. R80 in the human extracellular domain is K in Condylura cristata ALK4 (SEQ ID NO: 415), indicating that basic residues are tolerated at this position, including R, K, and H. Y77 in the human extracellular domain is F in Sus scrofa ALK4 (SEQ ID NO: 419), indicating that aromatic residues are tolerated at this position, including F, W, and Y. P93 in the human extracellular domain is relatively poorly conserved, appearing as S in Erinaceus europaeus ALK4 (SEQ ID NO: 416) and N in Gallus gallus ALK4, thus essentially any amino acid should be tolerated at this position.

Moreover, ALK4 proteins have been characterized in the art in terms of 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]. In addition to the teachings herein, these references provide amply guidance for how to generate ALK4 variants that retain one or more normal activities (e.g., ligand-binding activity).

For example, a defining structural motif known as a three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine residues located at varying 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]. Accordingly, the core ligand-binding domains of human ALK4, as demarcated by the outermost of these conserved cysteines, corresponds to positions 34-101 of SEQ ID NO: 84 (ALK4 precursor). The structurally less-ordered amino acids flanking these cysteine-demarcated 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 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, or 25 residues at the C-terminus without necessarily altering ligand binding. Exemplary ALK4 extracellular domains for N-terminal and/or C-terminal truncation include SEQ ID NOs: 86, 87, and 422.

In certain embodiments, the disclosure relates to heteromultimers that comprise at least one ALK4 polypeptide, which includes fragments, functional variants, and modified forms thereof. Preferably, ALK4 polypeptides for use as disclosed herein (e.g., heteromultimers comprising an ALK4 polypeptide and uses thereof) are soluble (e.g., an extracellular domain of ALK4). In other preferred embodiments, ALK4 polypeptides for use as disclosed herein bind to and/or inhibit (antagonize) activity (e.g., induction of Smad signaling) of one or more TGF-beta 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 NO: 84, 85, 86, 87, 88, 89, 92, 93, 421, and 422. In some embodiments, heteromultimers of the disclosure consist 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 NO: 84, 85, 86, 87, 88, 89, 92, 93, 422.

In certain aspects, the disclosure relates to a heteromultimer that comprises an ALK4-Fc fusion polypeptide. 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 an amino acid sequence that begins at any one of amino acids 23-34 (e.g., amino acid residues 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) SEQ ID NO: 84, 85, or 421 and ends at 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, and 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%8, 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 NOs: 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 Nos: 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.

E) ALK7 Polypeptides

In certain aspects, the disclosure relates to ActRII-ALK4 antagonists comprising an ALK7 polypeptide, which includes fragments, functional variants, and modified forms thereof as well as uses thereof (e.g., of treating, preventing, or reducing the progression rate and/or severity of heart failure (HF) or one or more complications of HF). As used herein, the term “ALK7” refers to a family of activin receptor-like kinase-7 (ALK7) proteins from any species and variant polypeptides derived from such ALK7 proteins by mutagenesis or other modifications (including, e.g., mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ALK7 polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication Nos. WO/2016/164089 and WO/2016/164503, which are incorporated herein by reference in their entirety. Reference to ALK7 herein is understood to be a reference to any one of the currently identified forms. Members of the ALK7 family are generally all transmembrane polypeptides, composed of a ligand-binding extracellular domain with 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 in (SEQ ID NO: 120) below. Preferably, ALK7 polypeptides to be used in accordance with the methods of the disclosure are soluble. The term “soluble ALK7 polypeptide,” as used herein, includes any naturally occurring extracellular domain of an ALK7 polypeptide as well as any variants thereof (including mutants, fragments and peptidomimetic forms) that retain a useful activity. For example, the extracellular domain of an ALK7 polypeptide binds to a ligand and is generally soluble. Examples of soluble ALK7 polypeptides include an ALK7 extracellular domain (SEQ ID NO: 123) below. Other examples of soluble ALK7 polypeptides comprise a signal sequence in addition to the extracellular domain of an ALK7 polypeptide. The signal sequence can be a native signal sequence of an ALK7, or a signal sequence from another polypeptide, such as a tissue plasminogen activator (TPA) signal sequence or a honey bee melatin signal sequence. In some embodiments, ALK7 polypeptides inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ALK7 polypeptides bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). Various examples of methods and assays for determining the ability for an ALK7 polypeptide to bind to and/or inhibit activity of one or more ActRII-ALK4 ligands are disclosed herein or otherwise well known in the art, which can be readily used to determine if an ALK7 polypeptide has the desired binding and/or antagonistic activities. Numbering of amino acids for all ALK7-related polypeptides described herein is based on the numbering of the human ALK7 precursor protein sequence provided below (SEQ ID NO: 120), unless specifically designated otherwise.

Four naturally occurring isoforms of human ALK7 have been described. The sequence of human ALK7 isoform 1 precursor polypeptide (NCBI Ref Seq NP_660302.2) is as follows:

(SEQ ID NO: 120)
1   MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTC
    QTEGACWASV MLTNGKEQVI
61  KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP
    TASPNAPKLG PMELAIIITV
121 PVCLLSIAAM LTVWACQGRQ CSYRKKKRPN VEEPLSECNL
    VNAGKTLKDL IYDVTASGSG
181 SGLPLLVQRT IARTIVLQEI VGKGRFGEVW HGRWCGEDVA
    VKIFSSRDER SWFREAEIYQ
241 TVMLRHENIL GFIAADNKDN GTWTQLWLVS EYHEQGSLYD
    YLNRNIVTVA GMIKLALSIA
301 SGLAHLHMEI VGTQGKPAIA HRDIKSKNIL VKKCETCAIA
    DLGLAVKHDS ILNTIDIPQN
361 PKVGTKRYMA PEMLDDTMNV NIFESFKRAD IYSVGLVYWE
    IARRCSVGGI VEEYQLPYYD
421 MVPSDPSIEE MRKVVCDQKF RPSIPNQWQS CEALRVMGRI
    MRECWYANGA ARLTALRIKK
481 TISQLCVKED CKA

The signal peptide is indicated by a single underline and the extracellular domain is indicated in bold font.

A processed extracellular ALK7 isoform 1 polypeptide sequence is as follows:

(SEQ ID NO: 123)
ELSPGLKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELN
AQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPME

A nucleic acid sequence encoding human ALK7 isoform 1 precursor poly peptide is shown below in SEQ ID NO: 233, corresponding to nucleotides 244-1722 of GenBank Reference Sequence NM_145259.2. A nucleic acid sequence encoding the processed extracellular ALK7 polypeptide (isoform 1) is show in in SEQ ID NO: 234.

(SEQ ID NO: 233)
ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCGC
AGCGGCCGCC              GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTG
ATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTC
ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCC
AGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAA
CCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCA
ACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAGCTGGCCATCAT
TATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGCGATGCTGACAGTAT
GGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGACCAAAT
GTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAAAAACTCT
GAAAGATCTGATTTATGATGTGACCGCCTCTGGATCTGGCTCTGGTCTAC
CTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAGGAAATA
GTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGTGTGGGGA
AGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATCTTGGTTTC
GTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAAAACATCCTT
GGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGACTCAACTTTG
GCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGACTATTTGAATA
GAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCT
AGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTACACAAGGTAAACC
TGCTATTGCTCATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGT
GTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCA
ATACTGAACACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAG
GTATATGGCTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTG
AGTCCTTCAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAA
ATAGCCCGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCC
TTATTATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGG
TTGTTTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGT
TGTGAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGC
CAACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTC
AACTTTGTGTCAAAGAAGACTGCAAAGCC
(SEQ ID NO: 234)
GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAAA
CTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAACCA
ATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTGAAT
GCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAATGCTG
CTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGCATCAC
CAAATGCCCCAAAACTTGGACCCATGGAG

An amino acid sequence of an alternative isoform of human ALK7, isoform 2 (NCBI Ref Seq NP_001104501.1), is shown in its processed form as follows (SEQ ID NO: 124), where the extracellular domain is indicated in bold font.

(SEQ ID NO: 124)
1   MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTD
    FCNNITLHLP TASPNAPKLG
61  PMELAIIITV PVCLLSIAAM LTVWACQGRQ CSYRKKKRPN
    VEEPLSECNL VNAGKTLKDL
121 IYDVTASGSG SGLPLLVQRT IARTIVLQEI VGKGRFGEVW
    HGRWCGEDVA VKIFSSRDER
181 SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVS
    EYHEQGSLYD YLNRNIVTVA
241 GMIKLALSIA SGLAHLHMEI VGTQGKPAIA HRDIKSKNIL
    VKKCETCAIA DLGLAVKHDS
301 ILNTIDIPQN PKVGTKRYMA PEMLDDTMNV NIFESFKRAD
    IYSVGLVYWE IARRCSVGGI
361 VEEYQLPYYD MVPSDPSIEE MRKVVCDQKF RPSIPNQWQS
    CEALRVMGRI MRECWYANGA
421 ARLTALRIKK TISQLCVKED CKA

An amino acid sequence of the extracellular ALK7 polypeptide (isoform 2) is as follows:

(SEQ ID NO: 125)
MLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHL
PTASPNAPKLGPME.

A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 2) is shown below in SEQ ID NO: 235, corresponding to nucleotides 279-1607 of NCBI Reference Sequence NM_001111031.1. A nucleic acid sequence encoding an extracellular ALK7 polypeptide (isoform 2) is shown in SEQ ID NO: 236.

(SEQ ID NO: 235)
ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTC
CAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAA
AACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTT
CCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAGCTGGCCA
TCATTATTACTGTGCCTGTTTGCCTCCTGTCCATAGCTGCGATGCTGAC
AGTATGGGCATGCCAGGGTCGACAGTGCTCCTACAGGAAGAAAAAGAGA
CCAAATGTGGAGGAACCACTCTCTGAGTGCAATCTGGTAAATGCTGGAA
AAACTCTGAAAGATCTGATTTATGATGTGACCGCCTCTGGATCTGGCTC
TGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTT
CAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGAT
GGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAG
ATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACAT
GAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTT
GGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATA
TGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTG
GCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTG
GTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAA
TATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTG
GCTGTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATC
CTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATAC
AATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCT
GTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAA
TTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCC
CTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCA
AGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGA
GAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGC
TCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGC
AAAGCC
(SEQ ID NO: 236)
ATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTC
CAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAA
AACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTT
CCAACAGCATCACCAAATGCCCCAAAACTTGGACCCATGGAG

An amino acid sequence of an alternative human ALK7 precursor polypeptide, isoform 3 (NCBI Ref Seq NP_001104502.1), is shown as follows (SEQ ID NO: 121), where the signal peptide is indicated by a single underline.

(SEQ ID NO: 121)
1   MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVI
61  KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP TGLPLLVQRT IARTIVLQEI
121 VGKGRFGEVW HGRWCGEDVA VKIFSSRDER SWFREAEIYQ TVMLRHENIL GFIAADNKDN
181 GTWTQLWLVS EYHEQGSLYD YLNRNIVTVA GMIKLALSIA SGLAHLHMEI VGTQGKPAIA
241 HRDIKSKNIL VKKCETCAIA DLGLAVKHDS ILNTIDIPQN PKVGTKRYMA PEMLDDTMNV
301 NIFESFKRAD IYSVGLVYWE IARRCSVGGI VEEYQLPYYD MVPSDPSIEE MRKVVCDQKF
361 RPSIPNQWQS CEALRVMGRI MRECWYANGA ARLTALRIKK TISQLCVKED CKA

The amino acid sequence of a processed ALK7 polypeptide (isoform 3) is as follows (SEQ ID NO: 126). This isoform lacks a 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: 126 are predicted as described below.

(SEQ ID NO: 126)
1   ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVI KSCVSLPELN AQVFCHSSNN
61  VTKTECCFTD FCNNITLHLP TGLPLLVQRT IARTIVLQEI VGKGRFGEVW HGRWCGEDVA
121 VKIFSSRDER SWFREAEIYQ TVMLRHENIL GFIAADNKDN GTWTQLWLVS EYHEQGSLYD
181 YLNRNIVTVA GMIKLALSIA SGLAHLHMEI VGTQGKPAIA HRDIKSKNIL VKKCETCAIA
241 DLGLAVKHDS ILNTIDIPQN PKVGTKRYMA PEMLDDTMNV NIFESFKRAD IYSVGLVYWE
301 IARRCSVGGI VEEYQLPYYD MVPSDPSIEE MRKVVCDQKF RPSIPNQWQS CEALRVMGRI
361 MRECWYANGA ARLTALRIKK TISQLCVKED CKA

A nucleic acid sequence encoding an unprocessed ALK7 polypeptide precursor polypeptide (isoform 3) is shown in SEQ ID NO: 237, corresponding to nucleotides 244-1482 of NCBI Reference Sequence NM_001111032.1. A nucleic acid sequence encoding a processed ALK7 polypeptide (isoform 3) is shown in SEQ ID NO: 238.

(SEQ ID NO: 237)
ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCG
CAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTG
TGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCA
GTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCC
TTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTAC
CAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCAC
CTTCCAACAGGTCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGA
TTGTGCTTCAGGAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCA
TGGAAGATGGTGTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGA
GATGAAAGATCTTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGC
TGCGACATGAAAACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAA
TGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGC
TCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGA
TCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGA
GATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAA
TCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACT
TAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACATACC
TCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTT
GATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACA
TCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGT
CGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCT
TCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGT
TTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGT
CATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGC
CTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAG
AAGACTGCAAAGCC
(SEQ ID NO: 238)
GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAA
ACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAAC
CAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTG
AATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAAT
GCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGG
TCTACCTCTGTTGGTTCAAAGGACAATTGCAAGGACGATTGTGCTTCAG
GAAATAGTAGGAAAAGGTAGATTTGGTGAGGTGTGGCATGGAAGATGGT
GTGGGGAAGATGTGGCTGTGAAAATATTCTCCTCCAGAGATGAAAGATC
TTGGTTTCGTGAGGCAGAAATTTACCAGACGGTCATGCTGCGACATGAA
AACATCCTTGGTTTCATTGCTGCTGACAACAAAGATAATGGAACTTGGA
CTCAACTTTGGCTGGTATCTGAATATCATGAACAGGGCTCCTTATATGA
CTATTTGAATAGAAATATAGTGACCGTGGCTGGAATGATCAAGCTGGCG
CTCTCAATTGCTAGTGGTCTGGCACACCTTCATATGGAGATTGTTGGTA
CACAAGGTAAACCTGCTATTGCTCATCGAGACATAAAATCAAAGAATAT
CTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGGACTTAGGGTTGGCT
GTGAAGCATGATTCAATACTGAACACTATCGACATACCTCAGAATCCTA
AAGTGGGAACCAAGAGGTATATGGCTCCTGAAATGCTTGATGATACAAT
GAATGTGAATATCTTTGAGTCCTTCAAACGAGCTGACATCTATTCTGTT
GGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTCAGTCGGAGGAATTG
TTGAGGAGTACCAATTGCCTTATTATGACATGGTGCCTTCAGATCCCTC
GATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGAAGTTTCGACCAAGT
ATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCGAGTCATGGGGAGAA
TAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCCCGCCTAACTGCTCT
TCGTATTAAGAAGACTATATCTCAACTTTGTGTCAAAGAAGACTGCAAA
GCC

An amino acid sequence of an alternative human ALK7 precursor polypeptide, isoform 4 (NCBI Ref Seq NP_001104503.1), is shown as follows (SEQ ID NO: 122), where the signal peptide is indicated by a single underline.

(SEQ ID NO: 122)
1   MTRALCSALR QALLLLAAAA ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVI
61  KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP TDNGTWTQLW LVSEYHEQGS
121 LYDYLNRNIV TVAGMIKLAL SIASGLAHLH MEIVGTQGKP AIAHRDIKSK NILVKKCETC
181 AIADLGLAVK HDSILNTIDI PQNPKVGTKR YMAPEMLDDT MNVNIFESFK RADIYSVGLV
241 YWEIARRCSV GGIVEEYQLP YYDMVPSDPS IEEMRKVVCD QKFRPSIPNQ WQSCEALRVM
301 GRIMRECWYA NGAARLTALR IKKTISQLCV KEDCKA

An amino acid sequence of a processed ALK7 polypeptide (isoform 4) is as follows (SEQ ID NO: 127). Like ALK7 isoform 3, isoform 4 lacks a 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.

(SEQ ID NO: 127)
1   ELSPGLKCVC LLCDSSNFTC QTEGACWASV MLTNGKEQVI KSCVSLPELN AQVFCHSSNN
61  VTKTECCFTD FCNNITLHLP TDNGTWTQLW LVSEYHEQGS LYDYLNRNIV TVAGMIKLAL
121 SIASGLAHLH MEIVGTQGKP AIAHRDIKSK NILVKKCETC AIADLGLAVK HDSILNTIDI
181 PQNPKVGTKR YMAPEMLDDT MNVNIFESFK RADIYSVGLV YWEIARRCSV GGIVEEYQLP
240 YYDMVPSDPS IEEMRKVVCD QKFRPSIPNQ WQSCEALRVM GRIMRECWYA NGAARLTALR
301 IKKTISQLCV KEDCKA

A nucleic acid sequence encoding the unprocessed ALK7 polypeptide precursor polypeptide (isoform 4) is shown in SEQ ID NO: 239, corresponding to nucleotides 244-1244 of NCBI Reference Sequence NM_001111033.1. A nucleic acid sequence encoding the processed ALK7 polypeptide (isoform 4) is shown in SEQ ID NO: 240.

(SEQ ID NO: 239)
ATGACCCGGGCGCTCTGCTCAGCGCTCCGCCAGGCTCTCCTGCTGCTCG
CAGCGGCCGCCGAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTG
TGATTCTTCAAACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCA
GTCATGCTAACCAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCC
TTCCAGAACTGAATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTAC
CAAAACCGAATGCTGCTTCACAGATTTTTGCAACAACATAACACTGCAC
CTTCCAACAGATAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAAT
ATCATGAACAGGGCTCCTTATATGACTATTTGAATAGAAATATAGTGAC
CGTGGCTGGAATGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCA
CACCTTCATATGGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTC
ATCGAGACATAAAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTG
TGCCATAGCGGACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAAC
ACTATCGACATACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGG
CTCCTGAAATGCTTGATGATACAATGAATGTGAATATCTTTGAGTCCTT
CAAACGAGCTGACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCC
CGGAGGTGTTCAGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATT
ATGACATGGTGCCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGT
TTGTGACCAGAAGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGT
GAAGCACTCCGAGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCA
ACGGAGCGGCCCGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCA
ACTTTGTGTCAAAGAAGACTGCAAAGCCTAA
(SEQ ID NO: 240)
GAGCTCTCGCCAGGACTGAAGTGTGTATGTCTTTTGTGTGATTCTTCAA
ACTTTACCTGCCAAACAGAAGGAGCATGTTGGGCATCAGTCATGCTAAC
CAATGGAAAAGAGCAGGTGATCAAATCCTGTGTCTCCCTTCCAGAACTG
AATGCTCAAGTCTTCTGTCATAGTTCCAACAATGTTACCAAAACCGAAT
GCTGCTTCACAGATTTTTGCAACAACATAACACTGCACCTTCCAACAGA
TAATGGAACTTGGACTCAACTTTGGCTGGTATCTGAATATCATGAACAG
GGCTCCTTATATGACTATTTGAATAGAAATATAGTGACCGTGGCTGGAA
TGATCAAGCTGGCGCTCTCAATTGCTAGTGGTCTGGCACACCTTCATAT
GGAGATTGTTGGTACACAAGGTAAACCTGCTATTGCTCATCGAGACATA
AAATCAAAGAATATCTTAGTGAAAAAGTGTGAAACTTGTGCCATAGCGG
ACTTAGGGTTGGCTGTGAAGCATGATTCAATACTGAACACTATCGACAT
ACCTCAGAATCCTAAAGTGGGAACCAAGAGGTATATGGCTCCTGAAATG
CTTGATGATACAATGAATGTGAATATCTTTGAGTCCTTCAAACGAGCTG
ACATCTATTCTGTTGGTCTGGTTTACTGGGAAATAGCCCGGAGGTGTTC
AGTCGGAGGAATTGTTGAGGAGTACCAATTGCCTTATTATGACATGGTG
CCTTCAGATCCCTCGATAGAGGAAATGAGAAAGGTTGTTTGTGACCAGA
AGTTTCGACCAAGTATCCCAAACCAGTGGCAAAGTTGTGAAGCACTCCG
AGTCATGGGGAGAATAATGCGTGAGTGTTGGTATGCCAACGGAGCGGCC
CGCCTAACTGCTCTTCGTATTAAGAAGACTATATCTCAACTTTGTGTCA
AAGAAGACTGCAAAGCCTAA

Based on the signal sequence of full-length ALK7 (isoform 1) in the rat (see NCBI Reference Sequence NP_620790.1) and on the high degree of sequence identity between human and rat ALK7, it is predicted that a processed form of human ALK7 isoform 1 is as follows (SEQ ID NO: 128).

(SEQ ID NO: 128)
1  LKCVCLLCDS SNFTCQTEGA CWASVMLING KEQVIKSCVS LPELNAQVFC HSSNNVTKTE
61 CCFTDFCNNI TLHLPTASPN APKLGPME

Active variants of processed ALK7 isoform 1 are predicted in which SEQ ID NO: 123 is truncated by 1, 2, 3, 4, 5, 6, or 7 amino acids at the N-terminus and SEQ ID NO: 128 is truncated by 1 or 2 amino acids at the N-terminus. Consistent with SEQ ID NO: 128, it is further expected that leucine is the N-terminal amino acid in the processed forms of human ALK7 isoform 3 (SEQ ID NO: 126) and human ALK7 isoform 4 (SEQ ID NO: 127).

In certain embodiments, the disclosure relates to heteromultimers that comprise at least one ALK7 polypeptide, which includes fragments, functional variants, and modified forms thereof. Preferably, ALK7 polypeptides for use in accordance with inventions of the disclosure (e.g., heteromultimers comprising an ALK7 polypeptide and uses thereof) are soluble (e.g., an extracellular domain of ALK7). In other preferred embodiments, ALK7 polypeptides for use in accordance with the disclosure bind to one or more ActRII-ALK4 ligand. Therefore, in some preferred embodiments, ALK7 polypeptides for use in accordance with the disclosure inhibit (antagonize) activity (e.g., induction of Smad signaling) of one or more ActRII-ALK4 ligands. In some embodiments, heteromultimers of the 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 NO:120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 255, 133, and 134. In some embodiments, heteromultimer of the disclosure consist 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 NO:120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 255, 133, and 134.

ALK7 is well-conserved among vertebrates, with large stretches of the extracellular domain completely conserved. For example, FIG. 22 depicts a multi-sequence alignment of a human ALK7 extracellular domain compared to various ALK7 orthologs. Accordingly, from these alignments, it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ALK7-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ALK7-ligand binding activities. Therefore, an active, human ALK7 variant polypeptide useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ALK7, or may include a residue that is similar to that in the human or other vertebrate sequences. Without meaning to be limiting, the following examples illustrate this approach to defining an active ALK7 variant. V61 in the human ALK7 extracellular domain (SEQ ID NO: 425) is isoleucine in Callithrix jacchus ALK7 (SEQ ID NO: 428), and so the position may be altered, and optionally may be altered to another hydrophobic residue such as L, I, or F, or a non-polar residue such as A. L32 in the human extracellular domain is R in Tarsius syrichta (SEQ ID NO: 429) ALK7, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y, and probably a non-polar residue such as A. K37 in the human extracellular domain is R in Pan troglodytes ALK7 (SEQ ID NO: 426), indicating that basic residues are tolerated at this position, including R, K, and H. P4 in the human extracellular domain is relatively poorly conserved, appearing as A in Pan troglodytes ALK7 thus indicating that a wide variety of amino acid should be tolerated at this position.

Moreover, ALK7 proteins have been characterized in the art in terms of structural and functional characteristics [e.g., Romano et al (2012) Journal of Molecular Modeling 18(8): 3617-3625]. For example, a defining structural motif known as a three-finger toxin fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine residues located at varying 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]. Accordingly, the core ligand-binding domains of human ALK7, as demarcated by the outermost of these conserved cysteines, corresponds to positions 28-92 of SEQ ID NO: 120. The structurally less-ordered amino acids flanking these cysteine-demarcated 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 by 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-terminal and/or C-terminal truncation include SEQ ID NOs: 123, 125, 126, and 127.

Accordingly, a general formula for an active portion (e.g., a ligand-binding portion) of ALK7 comprises amino acids 28-92 of SEQ ID NO: 120. Therefore ALK7 polypeptides may, for example, comprise, consists essentially of, or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%8, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ALK7 beginning at a residue corresponding to any one of amino acids 20-28 (e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, or 28) of SEQ ID NO: 120 and ending at a position corresponding to any one amino acids 92-113 (e.g., ending at any one of amino acids 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113) of SEQ ID NO: 120.

Other examples include constructs that begin at a position from 21-28 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, or 28), 24-28 (e.g., any one of positions 24, 25, 26, 27, or 28), or 25-28 (e.g., any one of positions 25, 26, 27, or 28) of SEQ ID NO: 120 and end at a position from 93-112 (e.g., any one of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112), 93-110 (e.g., any one of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110), 93-100 (e.g., any one of positions 93, 94, 95, 96, 97, 98, 99, or 100), or 93-95 (e.g., any one of positions 93, 94, or 95) of SEQ ID NO: 120. Variants within these ranges are also contemplated, particularly those 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.

The variations described herein may be combined in various ways. In some embodiments, ALK7 variants comprise 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, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain (as noted above).

F) Follistatin Polypeptides

In other aspects, an ActRII-ALK4 antagonist is a follistatin polypeptide. As described herein, follistatin polypeptides may be used treat, prevent, or reduce the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies), particularly treating, preventing or reducing the progression rate and/or severity of one or more heart failure-associated complications.

The term “follistatin polypeptide” includes polypeptides comprising any naturally occurring polypeptide of follistatin as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity, and further includes any functional monomer or multimer of follistatin. In certain preferred embodiments, follistatin polypeptides of the disclosure bind to and/or inhibit activin activity, particularly activin A. Variants of follistatin polypeptides that retain activin binding properties can be identified based on previous studies involving follistatin and activin interactions. For example, WO2008/030367 discloses specific follistatin domains (“FSDs”) that are shown to be important for activin binding. As shown below in SEQ ID NOs: 392-394, the follistatin N-terminal domain (“FSND” SEQ ID NO: 392), FSD2 (SEQ ID NO: 394), and to a lesser extent FSD1 (SEQ ID NO: 393) represent exemplary domains within follistatin that are important for activin binding. In addition, methods for making and testing libraries of polypeptides are described above in the context of ActRII polypeptides, and such methods also pertain to making and testing variants of follistatin. Follistatin polypeptides include polypeptides derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and optionally at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. Examples of follistatin polypeptides include the mature follistatin polypeptide or shorter isoforms or other variants of the human follistatin precursor polypeptide (SEQ ID NO: 390) as described, for example, in WO2005/025601.

The human follistatin precursor polypeptide isoform FST344 is as follows:

(SEQ ID NO: 390; NCBI Reference No. NP_037541.1)
1   MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL
51  SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC
101 GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC
151 KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA
201 SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC
251 TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA
301 ACSSGVLLEV KHSGSCNSIS EDTEEEEEDE DQDYSFPISS ILEW

The signal peptide is underlined; also underlined above are the last 27 residues which represent the C-terminal extension distinguishing this follistatin isoform from the shorter follistatin isoform FST317 shown below.

The human follistatin precursor polypeptide isoform FST317 is as follows:

(SEQ ID NO: 391; NCBI Reference No. NP_006341.1)
1   MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL
51  SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC
101 GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC
151 KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA
201 SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC
251 TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA
301 ACSSGVLLEV KHSGSCN

The signal peptide is underlined.

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

(SEQ ID NO: 392; FSND)
GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDN
TLFKWMIFNGGAPNCIPCK

The FSD1 and FSD2 sequences are as follows:

(SEQ ID NO: 393; FSD1)
ETCENVDCGPGKKCRMNKKNKPRCV
(SEQ ID NO: 394; FSD2)
KTCRDVFCPGSSTCVVDQTNNAYCVT

G) Fusion Polypeptides

In certain aspects, the disclosure provides for ActRII-ALK4 antagonists that are fusion polypeptides. The fusion polypeptides may be prepared according to any of the methods disclosed herein or that are known in the art.

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 portions 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 may be arranged in a manner as follows (N-terminus to C-terminus): A-B-C or C-B-A. In such embodiments, the fusion polypeptide may be arranged in a manner as follows (N-terminus to C-terminus): X-A-B-C or X-C-B-A. In some embodiments, the fusion polypeptide comprises each of A, B and C (and optionally a leader sequence), and comprises no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 additional amino acids (but which may include further post-translational modifications, such as glycosylation).

In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and 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 positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and 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 positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises an alanine between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises an alanine between X and C. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises a glycine and an alanine between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises a glycine and an alanine between X and C. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises a threonine between X and A. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises a threonine between X and C. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-A-B-C, and the fusion polypeptide comprises a threonine between A and B. In some embodiments, the fusion polypeptide comprises a leader sequence positioned in a manner as follows (N-terminus to C-terminus): X-C-B-A, and the fusion polypeptide comprises a threonine between C and B.

In certain aspects, fusion proteins of the disclosure comprise at least a portion of an ActRII-ALK4 ligand trap (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) and one or more heterologous portions (e.g., an immunoglobulin Fc domain), optionally with one or more linker domain sequence positioned between the ActRII-ALK4 ligand trap domain and the one or more heterologous portions. Well-known examples of such heterologous portions include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.

A heterologous portion may be selected so as to confer a desired property. For example, some heterologous portions are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen) useful with (HIS6) fusion partners. As another example, a heterologous portion may be selected so as to facilitate detection of the fusion polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the heterologous portions have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the heterologous portion by subsequent chromatographic separation.

In certain preferred embodiments, an ActRII-ALK4 ligand trap domain (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) is fused, optionally with an intervening linker domain, to a heterologous domain that stabilizes the ActRII-ALK4 ligand trap domain in vivo (a “stabilizer” domain). In general, “stabilizing” is meant anything that increases serum half-life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect of the agent. Fusion polypeptides with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of heterologous portions that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains. In some embodiments, a stabilizing domain may also function as a multimerization domain such multifunctional domains include, for example, Fc immunoglobulin domains. Various examples of Fc immunoglobulin domains and Fc-fusion proteins comprising one or more ActRII-ALK4 ligand trap domain are described throughout the disclosure.

In some embodiments, fusion proteins of the disclosure may additionally include any of various leader sequences at the N-terminus. Such a sequence would allow the peptides to be expressed and targeted to the secretion pathway in a eukaryotic system. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783 (1992). Alternatively, a native signal sequence may be used to effect extrusion from the cell. Possible leader sequences include native leaders, tissue plasminogen activator (TPA) and honeybee mellitin (SEQ ID NOs. 379, 9, 8, and 7 respectively). Examples of fusion proteins incorporating a TPA leader sequence include SEQ ID NOs: 6, 31, 34, 37, 40, 43, 46, 49, 51, 88, 92, 129, 133, 247, 276, 279, 333, 336, 339, 342, 345, 348, 351, 354, 381, 396, 402, and 406. Processing of signal peptides may vary depending on the leader sequence chosen, the cell type used and culture conditions, among other variables, and therefore actual N-terminal start sites for mature (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) polypeptides may shift by 1, 2, 3, 4 or 5 amino acids in either the N-terminal or C-terminal direction.

Preferred fusion proteins comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 12, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 50, 51, 52, 54, 55, 88, 89, 92, 93, 129, 130, 133, 134, 247, 249, 276, 278, 279, 332, 333, 335, 336, 338, 339, 341, 342, 344, 345, 347, 348, 350, 351, 353, 354, 356, 378, 380, 381, 385, 396, 398, 401, 402, 403, 406, 408, and 409.

I. Multimerization Domains

In certain aspects embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) of the present disclosure comprise at least one multimerization domain. As disclosed herein, the term “multimerization domain” refers to an amino acid or sequence of amino acids that promote covalent or non-covalent interaction between at least a first polypeptide and at least a second polypeptide. Polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may be joined covalently or non-covalently to a multimerization domain. In some embodiments, a multimerization domain promotes interaction between a first polypeptide (e.g., ActRIIB or ActRIIA polypeptide) and a second polypeptide (e.g., an ALK4 polypeptide or an ALK7 polypeptide) to promote heteromultimer formation (e.g., heterodimer formation), and optionally hinders or otherwise disfavors homomultimer formation (e.g., homodimer formation), thereby increasing the yield of desired heteromultimer (see, e.g., FIG. 8B). In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may from heterodimers through covalent interactions. In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may from heterodimers through non-covalent interactions. In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may from heterodimers through both covalent and non-covalent interactions. In some embodiments, a multimerization domain promotes interaction between a first polypeptide and a second polypeptide to promote homomultimer formation, and optionally hinders or otherwise disfavors heteromultimer formation, thereby increasing the yield of desired homomultimer. In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) form homodimers. In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may from homodimers through covalent interactions. In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may from homodimers through non-covalent interactions. In some embodiments, polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) may from homodimers through both covalent and non-covalent interactions.

In certain aspects, a multimerization domain may comprise one component of an interaction pair. In some embodiments, the polypeptides disclosed herein may form polypeptide complexes comprising a first polypeptide covalently or non-covalently associated with a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of a first ActRII-ALK4 ligand trap polypeptide (e.g., a ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide) and the amino acid sequence of a first member of an interaction pair (e.g., a first immunoglobulin Fc domain); and the second polypeptide comprises the amino acid sequence of a second ActRII-ALK4 ligand trap polypeptide (e.g., a ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide), and the amino acid sequence of a second member of an interaction pair (e.g., a second immunoglobulin Fc domain). In some embodiments, the polypeptides disclosed herein may form polypeptide complexes comprising a first polypeptide covalently or non-covalently associated with a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of an ActRIIA polypeptide and the amino acid sequence of a first member of an interaction pair; and the second polypeptide comprises the amino acid sequence of an ALK4 polypeptide or an ALK7 polypeptide, and the amino acid sequence of a second member of an interaction pair. In some embodiments, the polypeptides disclosed herein may form polypeptide complexes comprising a first polypeptide covalently or non-covalently associated with a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of an ActRIIB polypeptide and the amino acid sequence of a first member of an interaction pair; and the second polypeptide comprises the amino acid sequence of an ALK4 polypeptide or an ALK7 polypeptide, and the amino acid sequence of a second member of an interaction pair. In some embodiments, the interaction pair may be any two polypeptide sequences that interact to form a dimeric complex, either a heterodimeric or homodimeric complex. An interaction pair may be selected to confer an improved property/activity such as increased serum half-life, or to act as an adaptor on to which another moiety is attached to provide an improved property/activity. For example, a polyethylene glycol or glycosylation moiety may be attached to one or both components of an interaction pair to provide an improved property/activity such as improved serum half-life.

The first and second members of the interaction pair may be an asymmetric pair, meaning that the members of the pair preferentially associate with each other rather than self-associate. Accordingly, first and second members of an asymmetric interaction pair may associate to form a heterodimeric complex (see, e.g., FIG. 8B). Alternatively, the interaction pair may be unguided, meaning that the members of the pair may associate with each other or self-associate without substantial preference and thus may have the same or different amino acid sequences (see, e.g., FIG. 8A). Accordingly, first and second members of an unguided interaction pair may associate to form a homodimer complex or a heterodimeric complex. Optionally, the first member of the interaction pair (e.g., an asymmetric pair or an unguided interaction pair) associates covalently with the second member of the interaction pair. Optionally, the first member of the interaction pair (e.g., an asymmetric pair or an unguided interaction pair) associates non-covalently with the second member of the interaction pair. In certain preferred embodiments, polypeptides disclosed herein form heterodimeric or homodimeric complexes, although higher order heteromultimeric and homomultimeric complexes are also included such as, but not limited to, heterotrimers, homotrimers, heterotetramers, homotetramers, and further oligomeric structures (see, e.g., FIG. 11-13, which may also be applied to both ActRII-ALK4 and ActRII-ALK7 oligomeric structures).

Ia Fc-fusion Proteins

As specific examples of fusion polypeptides comprising a multimerization domain, the disclosure provides fusion polypeptides comprising an ActRII-ALK4 ligand trap polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide) fused to a polypeptide comprising a constant domain of an immunoglobulin, such as a CH1, CH2, or CH3 domain of an immunoglobulin or an immunoglobulin Fc domain. As used herein, the term “immunoglobulin Fc domain” or simply “Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain. In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region. In some embodiments, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). In certain preferred embodiments, the constant region is derived from IgG1. Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ), may be used. The choice of appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087 and 5,726,044, which is incorporated herein in its entirety. The choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art. In some embodiments, portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CH3 domain of Fc gamma or the homologous domains in any of IgA, IgD, IgE, or IgM. Furthermore, it is contemplated that substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed herein. One example would be to introduce amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for Fc receptors (Cole et al. (1997) J. Immunol. 159:3613). Fc domains derived from human IgG1, IgG2, IgG3, and IgG4 are provided herein.

An example of a native amino acid sequence that may be used for the Fc portion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 13). Dotted underline indicates the hinge region, and solid underline indicates positions with naturally occurring variants. In part, the disclosure provides polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) comprising, consisting of, or consisting essentially of an amino acid sequence with 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13.

Naturally occurring variants in G1Fc would include E134D and M136L according to the numbering system used in SEQ ID NO: 13 (see Uniprot P01857).

(SEQ ID NO: 13)
1
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTOKSLS LSPGK

In some embodiments, the disclosure provides Fc fusion polypeptides comprising an ActRII-ALK4 ligand trap polypeptide domain (e.g., an ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide domain), including variants as well as homomultimers (e.g., homodimers) and heteromultimers (e.g., heterodimers including, for example, ActRIIA:ALK4, ActRIIB:ALK4, ActRIIA:ALK7, and ActRIIB:ALK7 heterodimers) thereof, fused to one or more Fc polypeptide domains that are 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.

An example of a native amino acid sequence that may be used for the Fc portion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 14). Dotted underline indicates the hinge region and double underline indicates positions where there are data base conflicts in the sequence (according to UniProt P01859). In part, the disclosure provides polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) comprising, consisting of, or consisting essentially of an amino acid sequence with 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14.

(SEQ ID NO: 14)
1
51  FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS
101 NKGLPAPIEK TISKTKGQPR EPQVYTIPPS REEMTKNQVS LTCLVKGFYP
151 SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVES
201 CSVMHEALHN HYTQKSLSLS PGK

In some embodiments, the disclosure provides Fc fusion polypeptides comprising an ActRII-ALK4 ligand trap polypeptide domain (e.g., an ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide domain), including variants as well as homomultimers (e.g., homodimers) and heteromultimers (e.g., heterodimers including, for example, ActRIIA:ALK4, ActRIIB:ALK4, ActRIIA:ALK7, and ActRIIB:ALK7 heterodimers) thereof, fused to one or more Fc polypeptide domains that are 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.

Two examples of amino acid sequences that may be used for the Fc portion of human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be up to four times as long as in other Fc chains and contains three identical 15-residue segments preceded by a similar 17-residue segment. The first G3Fc sequence shown below (SEQ ID NO: 15) contains a short hinge region consisting of a single 15-residue segment, whereas the second G3Fc sequence (SEQ ID NO: 16) contains a full-length hinge region. In each case, dotted underline indicates the hinge region, and solid underline indicates positions with naturally occurring variants according to UniProt P01859. In part, the disclosure provides polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) comprising, consisting of, or consisting essentially of an amino acid sequence with 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 15. In part, the disclosure provides polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) comprising, consisting of, or consisting essentially of an amino acid sequence with 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 16.

(SEQ ID NO: 15)
1
51  VSHEDPEVQF KWYVDGVEVH NAKTKPREEQ YNSTFRVVSV LTVLHQDWLN
101 GKEYKCKVSN KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL
151 TCLVKGFYPS DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS
201 RWQQGNIFSC SVMHEALHNR FTQKSLSLSP GK
(SEQ ID NO: 16)
1
51
101 EDPEVQFKWY VDGVEVHNAK TKPREEQYNS TFRVVSVLIV LHQDWLNGKE
151 YKCKVSNKAL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL
201 VKGFYPSDIA VEWESSGQPE NNYNTTPPML DSDGSFFLYS KLTVDKSRWQ
251 QGNIFSCSVM HEALHNRFTQ KSLSLSPGK

Naturally occurring variants in G3Fc (for example, see Uniprot P01860) include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del, F221Y when converted to the numbering system used in SEQ ID NO: 15, and the present disclosure provides fusion polypeptides comprising G3Fc domains containing one or more of these variations. In addition, the human immunoglobulin IgG3 gene (IGHG3) shows a structural polymorphism characterized by different hinge lengths [see Uniprot P01859]. Specifically, variant WIS is lacking most of the V region and all of the CH1 region. It has an extra interchain disulfide bond at position 7 in addition to the 11 normally present in the hinge region. Variant ZUC lacks most of the V region, all of the CH1 region, and part of the hinge. Variant OMM may represent an allelic form or another gamma chain subclass. The present disclosure provides additional fusion polypeptides comprising G3Fc domains containing one or more of these variants.

In some embodiments, the disclosure provides Fc fusion polypeptides comprising an ActRII-ALK4 ligand trap polypeptide domain (e.g., an ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide domain), including variants as well as homomultimers (e.g., homodimers) and heteromultimers (e.g., heterodimers including, for example, ActRIIA:ALK4, ActRIIB:ALK4, ActRIIA:ALK7, and ActRIIB:ALK7 heterodimers) thereof, fused to one or more Fc polypeptide domains that are 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.

In some embodiments, the disclosure provides Fc fusion polypeptides comprising an ActRII-ALK4 ligand trap polypeptide domain (e.g., an ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide domain), including variants as well as homomultimers (e.g., homodimers) and heteromultimers (e.g., heterodimers including, for example, ActRIIA:ALK4, ActRIIB:ALK4, ActRIIA:ALK7, and ActRIIB:ALK7 heterodimers) thereof, fused to one or more Fc polypeptide domains that are 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.

An example of a native amino acid sequence that may be used for the Fc portion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 17). Dotted underline indicates the hinge region. In part, the disclosure provides polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptides) comprising, consisting of, or consisting essentially of an amino acid sequence with 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17.

(SEQ ID NO: 17)
1
51  EDPEVQFNWY VDGVEVHNAK TKPREEQENS TYRVVSVLTV LHQDWLNGKE
101 YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL
151 VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ
201 EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

In some embodiments, the disclosure provides Fc fusion polypeptides comprising an ActRII-ALK4 ligand trap polypeptide domain (e.g., an ActRIIA, ActRIIB, ALK4, ALK4, and follistatin polypeptide domain), including variants as well as homomultimers (e.g., homodimers) and heteromultimers (e.g., heterodimers including, for example, ActRIIA:ALK4, ActRIIB:ALK4, ActRIIA:ALK7, and ActRIIB:ALK7 heterodimers) thereof, fused to one or more Fc polypeptide domains that are 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.

A variety of engineered mutations in the Fc domain are presented herein with respect to the G1Fc sequence (SEQ ID NO: 13), and analogous mutations in G2Fc, G3Fc, and G4Fc can be derived from their alignment with G1Fc in FIG. 7. Due to unequal hinge lengths, analogous Fc positions based on isotype alignment (FIG. 7) possess different amino acid numbers in SEQ ID NOs: 13, 14, 15, and 17. It can also be appreciated that a given amino acid position in an immunoglobulin sequence consisting of hinge, C_H2, and C_H3 regions (e.g., SEQ ID NOs: 13, 14, 15, 16, or 17) will be identified by a different number than the same position when numbering encompasses the entire IgG1 heavy-chain constant domain (consisting of the CH1, hinge, CH2, and CH3 regions) as in the Uniprot database. For example, correspondence between selected CH3 positions in a human G1Fc sequence (SEQ ID NO: 13), the human IgG1 heavy chain constant domain (Uniprot P01857), and the human IgG1 heavy chain is as follows.

Correspondence of CH3 Positions in Different Numbering Systems
G1Fc
(Numbering	IgG1 heavy chain
begins at first	constant domain	IgG1 heavy chain
threonine in	(Numbering	(EU numbering scheme
hinge region)	begins at CH1)	of Kabat et al., 1991*)
Y127	Y232	Y349
S132	S237	S354
E134	E239	E356
K138	K243	K360
T144	T249	T366
L146	L251	L368
N162	N267	N384
K170	K275	K392
D177	D282	D399
D179	D284	D401
Y185	Y290	Y407
K187	K292	K409
H213	H318	H435
K217	K322	K439
* Kabat et al. (eds) 1991; pp. 688-696 in SequencesofProteinsofImmunologicalInterest, 5th ed., Vol. 1, NIH, Bethesda, MD.

In some embodiments, the disclosure provides antibodies and Fc fusion proteins with engineered or variant Fc regions. Such antibodies and Fc fusion proteins may be useful, for example, in modulating effector functions, such as, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Additionally, the modifications may improve the stability of the antibodies and Fc fusion proteins. Amino acid sequence variants of the antibodies and Fc fusion proteins are prepared by introducing appropriate nucleotide changes into the DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies and Fc fusion proteins disclosed herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antibodies and Fc fusion proteins, such as changing the number or position of glycosylation sites.

Antibodies and Fc fusion proteins with reduced effector function may be produced by introducing changes in the amino acid sequence, including, but are not limited to, the Ala-Ala mutation described by Bluestone et al. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 Cell Immunol 200; 16-26). Thus, in certain embodiments, Fc fusion proteins of the disclosure with mutations within the constant region including the Ala-Ala mutation may be used to reduce or abolish effector function. According to these embodiments, antibodies and Fc fusion proteins may comprise a mutation to an alanine at position 234 or a mutation to an alanine at position 235, or a combination thereof. In one embodiment, the antibody or Fc fusion protein comprises an IgG4 framework, wherein the Ala-Ala mutation would describe a mutation(s) from phenylalanine to alanine at position 234 and/or a mutation from leucine to alanine at position 235. In another embodiment, the antibody or Fc fusion protein comprises an IgG1 framework, wherein the Ala-Ala mutation would describe a mutation(s) from leucine to alanine at position 234 and/or a mutation from leucine to alanine at position 235. While alanine substitutions at these sites are effective in reducing ADCC in both human and murine antibodies, these substitutions are less effective at reducing CDC activity. Another single variant P329A, identified by a random mutagenesis approach to map the Clq binding site of the Fc, is highly effective at reducing CDC activity while retaining ADCC activity. A combination of L234A, L235A, and P329A (LALA-PG, Kabat positions) substitutions have been shown to effectively silence the effector function of human IgG1 antibodies. For a detailed discussion of LALA, LALA-PG, and other mutations, see Lo et al. (2017) 1 Biol. Chem. 292:3900-3908, the contents of which are hereby incorporated herein by reference in their entirety. In some embodiments, Fc fusion proteins of the disclosure comprise L234A, L235A, and P329G mutations (LALA-PG; Kabat positions) in the Fc region of the heavy chain. The antibody or Fc fusion protein may alternatively or additionally carry other mutations, including the point mutation K322A in the CH2 domain (Hezareh et al. 2001 J Virol. 75: 12161-8).

In particular embodiments, the antibody or Fc fusion protein may be modified to either enhance or inhibit complement dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region (see, e.g., U.S. Pat. No. 6,194,551). Alternatively, or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody or Fc fusion protein thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature 322: 738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351.

Ib Heteromultimers

Many methods known in the art can be used to generate ActRIIB:ALK4 heteromultimers, ActRIIB:ALK7 heteromultimers, ActRIIA:ALK4 heteromultimers, and ActRIIA:ALK7 heteromultimers as disclosed herein. For example, non-naturally occurring disulfide bonds may be constructed by replacing on a first polypeptide (e.g., an ActRIIB or ActRIIA polypeptide) a naturally occurring amino acid with a free thiol-containing residue, such as cysteine, such that the free thiol interacts with another free thiol-containing residue on a second polypeptide (e.g., an ALK4 or ALK7 polypeptide) such that a disulfide bond is formed between the first and second polypeptides. Additional examples of interactions to promote heteromultimer formation include, but are not limited to, ionic interactions such as described in Kjaergaard et al., WO2007147901; electrostatic steering effects such as described in Kannan et al., U.S. Pat. No. 8,592,562; coiled-coil interactions such as described in Christensen et al., U.S. 20120302737; leucine zippers such as described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584; and helix-turn-helix motifs such as described in Pack et al., (1993) Bio/Technology 11: 1271-1277. Linkage of the various segments may be obtained via, e.g., covalent binding such as by chemical cross-linking, peptide linkers, disulfide bridges, etc., or affinity interactions such as by avidin-biotin or leucine zipper technology.

As specific examples, the present disclosure provides fusion proteins comprising ActRIIB, ActRIIA, ALK4, or ALK7 fused to a polypeptide comprising a constant domain of an immunoglobulin, such as a CH1, CH2, or CH3 domain derived from human IgG1, IgG2, IgG3, and/or IgG4 that has been modified to promote heteromultimer formation. A problem that arises in large-scale production of asymmetric immunoglobulin-based proteins from a single cell line is known as the “chain association issue”. As confronted prominently in the production of bispecific antibodies, the chain-association issue concerns the challenge of efficiently producing a desired multichain protein from among the multiple combinations that inherently result when different heavy chains and/or light chains are produced in a single cell line [see, for example, Klein et al (2012) mAbs 4:653-663]. This problem is most acute when two different heavy chains and two different light chains are produced in the same cell, in which case there are a total of 16 possible chain combinations (although some of these are identical) when only one is typically desired. Nevertheless, the same principle accounts for diminished yield of a desired multichain fusion protein that incorporates only two different (asymmetric) heavy chains.

Various methods are known in the art that increase desired pairing of Fc-containing fusion polypeptide chains in a single cell line to produce a preferred asymmetric fusion protein at acceptable yields [see, for example, Klein et al (2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology 67(2A): 95-106]. Methods to obtain desired pairing of Fc-containing chains include, but are not limited to, charge-based pairing (electrostatic steering), “knobs-into-holes” steric pairing, SEEDbody pairing, and leucine zipper-based pairing. See, for example, Ridgway et al (1996) Protein Eng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al (2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010); 285:19637-19646; Wranik et al (2012) J Biol Chem 287:43331-43339; U.S. Pat. No. 5,932,448; WO 1993/011162; WO 2009/089004, and WO 2011/034605. As described herein, these methods may be used to generate heterodimers comprising an ActRIIB polypeptide and another, optionally different, ActRIIB polypeptide, an ActRIIA polypeptide and another, optionally different, ActRIIA polypeptide, an ActRIIB polypeptide and an ActRIIA polypeptide, an ActRIIB polypeptide and an ALK4 polypeptide, an ActRIIB polypeptide and an ALK7 polypeptide, an ActRIIA polypeptide and an ALK4 polypeptide, or an ActRIIA polypeptide and an ALK7 polypeptide.

For example, one means by which interaction between specific polypeptides may be promoted is by engineering protuberance-into-cavity (knob-into-holes) complementary regions such as described in Arathoon et al., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., a first interaction pair) with larger side chains (e.g., tyrosine or tryptophan). Complementary “cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide (e.g., a second interaction pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Where a suitably positioned and dimensioned protuberance or cavity exists at the interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface.

At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged, and lysine, arginine, and histidine are positively charged. These charged residues can be used to promote heterodimer formation and at the same time hinder homodimer formation. Attractive interactions take place between opposite charges and repulsive interactions occur between like charges. In part, polypeptide complexes disclosed herein make use of the attractive interactions for promoting heteromultimer formation (e.g., heterodimer formation), and optionally repulsive interactions for hindering homodimer formation (e.g., homodimer formation) by carrying out site directed mutagenesis of charged interface residues.

For example, the IgG1 CH3 domain interface comprises four unique charge residue pairs involved in domain-domain interactions: Asp356-Lys439′, Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering in the second chain is indicated by (′)]. It should be noted that the numbering scheme used here to designate residues in the IgG1 CH3 domain conforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction will be represented twice in the structure (e.g., Asp-399-Lys409′ and Lys409-Asp399′). In the wild-type sequence, K409-D399′ favors both heterodimer and homodimer formation. A single mutation switching the charge polarity (e.g., K409E; positive to negative charge) in the first chain leads to unfavorable interactions for the formation of the first chain homodimer. The unfavorable interactions arise due to the repulsive interactions occurring between the same charges (negative-negative; K409E-D399′ and D399-K409E′). A similar mutation switching the charge polarity (D399K′; negative to positive) in the second chain leads to unfavorable interactions (K409′-D399K′ and D399K-K409′) for the second chain homodimer formation. But, at the same time, these two mutations (K409E and D399K′) lead to favorable interactions (K409E-D399K′ and D399-K409′) for the heterodimer formation.

The electrostatic steering effect on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360. The table below lists possible charge change mutations that can be used, alone or in combination, to enhance heteromultimer formation of the heteromultimers disclosed herein.

Examples of Pair-Wise Charged Residue Mutations to
Enhance Heterodimer Formation
			Interacting
			position	Corresponding
	Position in	Mutation in	in second	mutation in
	first chain	first chain	chain	second chain
	Lys409	Asp or Glu	Asp399′	Lys, Arg, or His
	Lys392	Asp or Glu	Asp399′	Lys, Arg, or His
	Lys439	Asp or Glu	Asp356′	Lys, Arg, or His
	Lys370	Asp or Glu	Glu357′	Lys, Arg, or His
	Asp399	Lys, Arg, or His	Lys409′	Asp or Glu
	Asp399	Lys, Arg, or His	Lys392′	Asp or Glu
	Asp356	Lys, Arg, or His	Lys439′	Asp or Glu
	Glu357	Lys, Arg, or His	Lys370′	Asp or Glu

In some embodiments, one or more residues that make up the CH3-CH3 interface in a fusion polypeptide of the instant application are replaced with a charged amino acid such that the interaction becomes electrostatically unfavorable. For example, a positive-charged amino acid in the interface (e.g., a lysine, arginine, or histidine) is replaced with a negatively charged amino acid (e.g., aspartic acid or glutamic acid). Alternatively, or in combination with the forgoing substitution, a negative-charged amino acid in the interface is replaced with a positive-charged amino acid. In certain embodiments, the amino acid is replaced with a non-naturally occurring amino acid having the desired charge characteristic. It should be noted that mutating negatively charged residues (Asp or Glu) to His will lead to increase in side chain volume, which may cause steric issues. Furthermore, His proton donor- and acceptor-form depends on the localized environment. These issues should be taken into consideration with the design strategy. Because the interface residues are highly conserved in human and mouse IgG subclasses, electrostatic steering effects disclosed herein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4. This strategy can also be extended to modifying uncharged residues to charged residues at the CH3 domain interface.

In certain aspects, the ActRII-ALK4 ligand trap to be used in accordance with the methods disclosed herein is a heteromultimer complex comprising at least one ALK polypeptide (e.g., an ALK4 or ALK7 polypeptide) associated, covalently or non-covalently, with at least one ActRII polypeptide (e.g., an ActRIIA or ActRIIB polypeptide). Preferably, polypeptides disclosed herein form heterodimeric complexes, although higher order heteromultimeric complexes (heteromultimers) are also included such as, but not limited to, heterotrimers, heterotetramers, and further oligomeric structures (see, e.g., FIGS. 11-13, which may also be applied to both ActRII-ALK4 and ActRII-ALK7 oligomeric structures). In some embodiments, ALK and/or ActRII polypeptides comprise at least one multimerization domain. Polypeptides disclosed herein may be joined covalently or non-covalently to a multimerization domain. Preferably, a multimerization domain promotes interaction between a first polypeptide (e.g., an ActRIIB or ActRIIA polypeptide) and a second polypeptide (e.g., an ALK4 or ALK7 polypeptide) to promote heteromultimer formation (e.g., heterodimer formation), and optionally hinders or otherwise disfavors homomultimer formation (e.g., homodimer formation), thereby increasing the yield of desired heteromultimer (see, e.g., FIG. 12).

In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered to be complementary on the basis of charge pairing (electrostatic steering). One of a pair of Fc sequences with electrostatic complementarity can be arbitrarily fused to an ActRIIB polypeptide, ActRIIA polypeptide, ALK4 polypeptide, or an ALK7 polypeptide of the construct, with or without an optional linker, to generate an ActRIIB-Fc, ActRIIA-Fc, ALK4-Fc, or ALK7-Fc fusion polypeptide. This single chain can be coexpressed in a cell of choice along with the Fc sequence complementary to the first Fc sequence to favor generation of the desired multichain construct (e.g., an ActRIIB-Fc-ALK4-Fc heteromultimer). In this example based on electrostatic steering, SEQ ID NO: 18 [human G1Fc(E134K/D177K)] and SEQ ID NO: 19 [human G1Fc(K170D/K187D)] are examples of complementary Fc sequences in which the engineered amino acid substitutions are double underlined, and an ActRIIB polypeptide, ActRIIA polypeptide, ALK4 polypeptide, or an ALK7 polypeptide of the construct can be fused to either SEQ ID NO: 18 or SEQ ID NO: 19, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 18 and 19).

(SEQ ID NO: 18)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRKEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLKSDG SFFLYSKLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK
(SEQ ID NO: 19)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYD TTPPVLDSDG SFFLYSDLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 19, 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: 18. In some embodiments, the disclosure relates to ActRIIB heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 18, 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: 19.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 19, 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: 18. In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 18, 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: 19.

In some embodiments, the disclosure relates to ActRIIA-ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 19, 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: 18. In some embodiments, the disclosure relates to ActRIIA heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 18, 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: 19.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 19, 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: 18. In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 18, 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: 19.

In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered for steric complementarity. In part, the disclosure provides knobs-into-holes pairing as an example of steric complementarity. One of a pair of Fe sequences with steric complementarity can be arbitrarily fused to an ActRIIB polypeptide, an ActRIIA polypeptide, an ALK4 polypeptide, or an ALK7 polypeptide of the construct, with or without an optional linker, to generate an ActRIIB-Fc, ActRIIA-Fe, ALK4-Fe, or ALK7-Fc fusion polypeptide. This single chain can be coexpressed in a cell of choice along with the Fc sequence complementary to the first Fc sequence to favor generation of the desired multichain construct. In this example based on knobs-into-holes pairing, SEQ ID NO: 20 [human G1Fc(T144Y)] and SEQ ID NO: 21 [human G1Fc(Y185T)] are examples of complementary Fc sequences in which the engineered amino acid substitutions are double underlined, and n ActRIIB polypeptide, ActRIIA polypeptide, ALK4 polypeptide, or ALK7 polypeptide of the construct can be fused to either SEQ ID NO: 20 or SEQ ID NO: 21, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 20 and 21).

(SEQ ID NO: 20)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLYCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK
(SEQ ID NO: 21)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLTSKLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 21, 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: 20. In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 20, 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: 21.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 21, 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: 20. In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 20, 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: 21.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 21, 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: 20. In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 20, 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: 21.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 21, 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: 20. In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 20, 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: 21.

An example of Fc complementarity based on knobs-into-holes pairing combined with an engineered disulfide bond is disclosed in SEQ ID NO: 22 [hG1Fc(S132C/T144W)] and SEQ ID NO: 23 [hG1Fc(Y127C/T144S/L146A/Y185V)]. The engineered amino acid substitutions in these sequences are double underlined, and an ActRIIB polypeptide, ActRIIA polypeptide, ALK4 polypeptide, or ALK7 polypeptide of the construct can be fused to either SEQ ID NO: 22 or SEQ ID NO: 23, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 22 and 23).

(SEQ ID NO: 22)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ
    VSLWCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK
(SEQ ID NO: 23)
1   THTCPPCPAP ELLGGPSVEL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ
    VSLSCAVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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, 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: 22. In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 22, 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.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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, 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: 22. In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 22, 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.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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, 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: 22. In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 22, 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.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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, 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: 22. In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 22, 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.

In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered to generate interdigitating R-strand segments of human IgG and IgA C_H3 domains. Such methods include the use of strand-exchange engineered domain (SEED) C_H3 heterodimers allowing the formation of SEEDbody fusion polypeptides [see, for example, Davis et al (2010) Protein Eng Design Sel 23:195-202]. One of a pair of Fc sequences with SEEDbody complementarity can be arbitrarily fused to a first ActRIIB polypeptide or second ActRIIB polypeptide of the construct, with or without an optional linker, to generate an ActRIIB-Fc fusion polypeptide. This single chain can be coexpressed in a cell of choice along with the Fc sequence complementary to the first Fc sequence to favor generation of the desired multichain construct. In this example based on SEEDbody (Sb) pairing, SEQ ID NO: 24 [hG1Fc(Sb_AG)] and SEQ ID NO: 25 [hG1Fc(Sb_GA)] are examples of complementary IgG Fc sequences in which the engineered amino acid substitutions from IgA Fc are double underlined, and a first ActRIIB polypeptide or second variant ActRIIB polypeptide, of the construct can be fused to either SEQ ID NO: 24 or SEQ ID NO: 25, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG1Fc, hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate an Fc monomer which may be used in the complementary IgG-IgA pair below (SEQ ID NOs: 24 and 25).

(SEQ ID NO: 24)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PFRPEVHLLP PSREEMTKNQ
    VSLTCLARGF
151 YPKDIAVEWE SNGQPENNYK TTPSRQEPSQ GTTTFAVTSK
    LTVDKSRWQQ
201 GNVFSCSVMH EALHNHYTQK TISLSPGK
(SEQ ID NO: 25)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PPSEELALNE
    LVTLTCLVKG
151 FYPSDIAVEW ESNGQELPRE KYLTWAPVLD SDGSFFLYSI
    LRVAAEDWKK
201 GDTFSCSVMH EALHNHYTQK SLDRSPGK

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 25, 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: 24. In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 24, 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: 25.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 25, 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: 24. In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 24, 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: 25.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 25, 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: 24. In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 24, 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: 25.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 25, 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: 24. In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 24, 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: 25.

In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains with a cleavable leucine zipper domain attached at the C-terminus of the Fc C_H3 domains. Attachment of a leucine zipper is sufficient to cause preferential assembly of heterodimeric antibody heavy chains. See, e.g., Wranik et al (2012) J Biol Chem 287:43331-43339. As disclosed herein, one of a pair of Fc sequences attached to a leucine zipper-forming strand can be arbitrarily fused to a first ActRIIB polypeptide or second ActRIIB polypeptide, of the construct, with or without an optional linker, to generate an ActRIIB-Fc fusion polypeptide. This single chain can be coexpressed in a cell of choice along with the Fc sequence attached to a complementary leucine zipper-forming strand to favor generation of the desired multichain construct. Proteolytic digestion of the construct with the bacterial endoproteinase Lys-C post purification can release the leucine zipper domain, resulting in an Fc construct whose structure is identical to that of native Fc. In this example based on leucine zipper pairing, SEQ ID NO: 26 [hG1Fc-Ap1 (acidic)] and SEQ ID NO: 27 [hG1Fc-Bp1 (basic)] are examples of complementary IgG Fc sequences in which the engineered complimentary leucine zipper sequences are underlined, and a ActRIIB polypeptide or second variant ActRIIB polypeptide of the construct can be fused to either SEQ ID NO: 26 or SEQ ID NO: 27, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that leucine zipper-forming sequences attached, with or without an optional linker, to hG1Fc, hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate an Fc monomer which may be used in the complementary leucine zipper-forming pair below (SEQ ID NOs: 26 and 27).

(SEQ ID NO: 26)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
    VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LEKELQALEK
    ENAQLEWELQ
251 ALEKELAQGA T
(SEQ ID NO: 27)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
    VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGKGGSAQ LKKKLQALKK
    KNAQLKWKLQ
251 ALKKKLAQGA T

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 27, 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: 26. In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 26, 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: 27.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 27, 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: 26. In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 26, 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: 27.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 27, 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: 26. In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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: 26, 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: 27.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 27, 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: 26. In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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: 26, 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: 27.

In part, the disclosure provides desired pairing of asymmetric Fc-containing polypeptide chains by methods described above in combination with additional mutations in the Fc domain which facilitate purification of the desired heteromeric species. An example uses complementarity of Fe domains based on knobs-into-holes pairing combined with an engineered disulfide bond, as disclosed in SEQ ID NOs: 22 and 23, plus additional substitution of two negatively charged amino acids (aspartic acid or glutamic acid) in one Fc-containing polypeptide chain and two positively charged amino acids (e.g., arginine) in the complementary Fc-containing polypeptide chain (SEQ ID NOs: 28-29). These four amino acid substitutions facilitate selective purification of the desired heteromeric fusion polypeptide from a heterogeneous polypeptide mixture based on differences in isoelectric point or net molecular charge. The engineered amino acid substitutions in these sequences are double underlined below, and an ActRIIB polypeptide, an ActRIIA polypeptide, an ALK4 polypeptide, or an ALK7 polypeptide of the construct can be fused to either SEQ ID NO: 28 or SEQ ID NO: 29, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair below (SEQ ID NOs: 28-29).

(SEQ ID NO: 28)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTENQ
    VSLWCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNHYTQDSLS LSPGK
(SEQ ID NO: 29)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ
    VSLSCAVKGF
151 YPSDIAVEWE SRGQPENNYK TTPPVLDSRG SFFLVSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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.

In some embodiments, the ActRIIB-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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.

Another example involves complementarity of Fc domains based on knobs-into-holes pairing combined with an engineered disulfide bond, as disclosed in SEQ ID NOs: 22-23, plus a histidine-to-arginine substitution at position 213 in one Fc-containing polypeptide chain (SEQ ID NO: 30). This substitution (denoted H435R in the numbering system of Kabat et al.) facilitates separation of desired heterodimer from undesirable homodimer based on differences in affinity for protein A. The engineered amino acid substitution is indicated by double underline, and an ActRIIB polypeptide, ActRIIA polypeptide, ALK4 polypeptide, or ALK7 polypeptide of the construct can be fused to either SEQ ID NO: 30 or SEQ ID NO: 23, but not both. Given the high degree of amino acid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that amino acid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 7) will generate complementary Fc pairs which may be used instead of the complementary hG1Fc pair of SEQ ID NO: 30 (below) and SEQ ID NO: 23.

(SEQ ID NO: 30)
1   THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
    VVDVSHEDPE
51  VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
    WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ
    VSLWCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
    DKSRWQQGNV
201 FSCSVMHEAL HNRYTQKSLS LSPGK

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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.

In some embodiments, 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. In some embodiments, the ALK4-Fc fusion polypeptide Fc domain comprises 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. In some embodiments, the ALK7-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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

In some embodiments, the ActRIIA-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

In some embodiments, the disclosure relates to ActRIIB:ALK4 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIB:ALK7 heteromultimer polypeptides comprising an ActRIIB-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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.

In some embodiments, the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185. In some embodiments, the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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.

In some embodiments, the ALK4-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185. In some embodiments, the ALK7-Fc fusion polypeptide Fc domain comprises a cysteine at amino acid position 132, glutamic acid at amino acid position 138, a tryptophan at amino acid position 144, and an aspartic acid at amino acid position 217. In some embodiments, 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, an arginine at amino acid position 162, an arginine at amino acid position 179, and a valine at amino acid position 185.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the ActRIIB-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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.

In some embodiments, 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. In some embodiments, the ALK4-Fc fusion polypeptide Fc domain comprises 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. In some embodiments, the ALK7-Fc fusion polypeptide Fc domain comprises 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.

In some embodiments, the disclosure relates to ActRIIA:ALK4 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK4-Fc fusion polypeptide wherein 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.

In some embodiments, the disclosure relates to ActRIIA:ALK7 heteromultimer polypeptides comprising an ActRIIA-Fc fusion polypeptide and an ALK7-Fc fusion polypeptide wherein 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

In some embodiments, 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. In some embodiments, 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. In some embodiments, the ActRIIA-Fc fusion polypeptide Fc domain comprises 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.

In certain embodiments, the disclosure relates to a heteromultimer comprising a first variant ActRIIB-Fc fusion polypeptide and a second variant ActRIIB-Fc fusion polypeptide, wherein the first variant ActRIIB polypeptide does not comprise the amino acid sequence of the second variant ActRIIB polypeptide. In some embodiments, an ActRIIB-Fc:ActRIIB-Fc heteromultimer binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, an ActRIIB-Fc:ActRIIB-Fc heteromultimer inhibits signaling of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, an ActRIIB-Fc:ActRIIB-Fc heteromultimer is a heterodimer.

In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one 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 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the one or more amino acid substitutions is 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. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one 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 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is 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. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation. In some embodiments, the heteromultimer is a heterodimer.

In certain aspects, the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide 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: 36, and second ActRIIB polypeptide 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, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide. In some embodiments, the first ActRIIB polypeptide comprises a glutamic acid at the amino acid position corresponding to 55 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide does not comprise a glutamic acid at the amino acid position corresponding to 55 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide comprises a lysine at the amino acid position corresponding to 55 of SEQ ID NO: 2. In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that promote heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation. In some embodiments, the heteromultimer is a heterodimer.

In certain aspects, the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide 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: 39, and second ActRIIB polypeptide 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, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide. In some embodiments, the first ActRIIB polypeptide comprises an isoleucine at the amino acid position corresponding to 82 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide does not comprise an isoleucine acid at the amino acid position corresponding to 82 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide comprises a phenylalanine at the amino acid position corresponding to 82 of SEQ ID NO: 2. In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that promote heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modification that inhibit heteromultimer formation. In some embodiments, the heteromultimer is a heterodimer.

In certain aspects, the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide 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, and second ActRIIB polypeptide 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, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide. In some embodiments, first ActRIIB polypeptide comprises a lysine at the amino acid position corresponding to 82 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide does not comprise a lysine acid at the amino acid position corresponding to 82 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide comprises a phenylalanine at the amino acid position corresponding to 82 of SEQ ID NO: 2. In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of L79, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, and D80 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79A, L79D, L79E, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, and D80R. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that promote heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that inhibit heteromultimer formation. In some embodiments, the heteromultimer is a heterodimer.

In certain aspects, the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide 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: 45, and second ActRIIB polypeptide 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: 48, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide. In some embodiments, the first ActRIIB polypeptide comprises an acidic amino acid position corresponding to 79 of SEQ ID NO: 2. In some embodiments, the acidic amino acid is an aspartic acid. In some embodiments, the acidic amino acid is a glutamic acid. In some embodiments, the second ActRIIB polypeptide does not comprise an acidic acid (e.g., aspartic acid or glutamic acid) at the amino acid position corresponding to 79 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide comprises a leucine at the amino acid position corresponding to 79 of SEQ ID NO: 2. In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that promote heteromultimer formation. In some embodiments, the first ActRIIB polypeptide and/or the second ActRIIB polypeptide comprise one or more amino acid modifications that inhibit heteromultimer formation. In some embodiments, the heteromultimer is a heterodimer.

In certain aspects, the disclosure relates to a heteromultimer comprising a first ActRIIB polypeptide 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: 50, and second ActRIIB polypeptide 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: 52, wherein the first ActRIIB polypeptide does not comprise the amino acid sequence of the second ActRIIB polypeptide. In some embodiments, the first ActRIIB polypeptide comprises an acidic amino acid position corresponding to 79 of SEQ ID NO: 2. In some embodiments, the acidic amino acid is an aspartic acid. In some embodiments, the acidic amino acid is a glutamic acid. In some embodiments, the second ActRIIB polypeptide does not comprise an acidic acid (e.g., aspartic acid or glutamic acid) at the amino acid position corresponding to 79 of SEQ ID NO: 2. In some embodiments, the second ActRIIB polypeptide comprises a leucine at the amino acid position corresponding to 79 of SEQ ID NO: 2. In some embodiments, the first ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, L79P, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A. In some embodiments, the second ActRIIB polypeptide comprises one or more amino acid substitutions at the amino acid positions corresponding to any one of F82, A24, K74, R64, P129, P130, E37, R40, D54, R56, W78, D80, and F82 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions is selected from the group consisting of: A24N, K74A, R64K, R64N, K74A, P129S, P130A, P130R, E37A, R40A, D54A, R56A, K74F, K74I, K74Y, W78A, D80A, D80F, D80G, D80I, D80K, D80M, D80M, D80N, D80R, and F82A.

In certain aspects, the present disclosure relates to heteromultimers comprising one or more ALK4 receptor polypeptides (e.g., SEQ ID Nos: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, 422 and variants thereof) and one or more ActRIIB receptor polypeptides (e.g., 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, 409 and variants thereof), including uses thereof (e.g. treating heart failure in a patient in need thereof), which are generally referred to herein as “ActRIIB:ALK4 heteromultimer” or “ActRIIB-ALK4 heteromultimers”, including uses thereof (e.g., treating heart failure in a patient in need thereof). Preferably, ActRIIB:ALK4 heteromultimers are soluble [e.g., a heteromultimer complex comprises a soluble portion (domain) of an ALK4 receptor and a soluble portion (domain) of an ActRIIB receptor]. In general, the extracellular domains of ALK4 and ActRIIB correspond to soluble portion of these receptors. Therefore, in some embodiments, ActRIIB:ALK4 heteromultimers comprise an extracellular domain of an ALK4 receptor and an extracellular domain of an ActRIIB receptor. In some embodiments, ActRIIB:ALK4 heteromultimers inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIB:ALK4 heteromultimers bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIB:ALK4 heteromultimers comprise at least one ALK4 polypeptide that comprises, consists essentially of, or consists of a 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: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, and 422. In some embodiments, ActRIIB:ALK4 heteromultimer complexes of the disclosure comprise at least one ALK4 polypeptide that comprises, consists essentially of, consists of a 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 a portion of ALK4 beginning at a residue corresponding to any one of amino acids 24-34, 25-34, or 26-34 of SEQ ID NO: 84 and ending at a position from 101-126, 102-126, 101-125, 101-124, 101-121, 111-126, 111-125, 111-124, 121-126, 121-125, 121-124, or 124-126 of SEQ ID NO: 84. In some embodiments, ActRIIB:ALK4 heteromultimers comprise at least one ALK4 polypeptide that comprises, consists essentially of, consists of a 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 amino acids 34-101 with respect to SEQ ID NO: 84. In some embodiments, ActRIIB-ALK4 heteromultimers comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 any one of SEQ ID NOs: 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, ActRIIB:ALK4 heteromultimer complexes of the disclosure comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 a portion of ActRIIB beginning at a residue corresponding to any one of amino acids 20-29, 20-24, 21-24, 22-25, or 21-29 and end at a position from 109-134, 119-134, 119-133, 129-134, or 129-133 of SEQ ID NO: 2. In some embodiments, ActRIIB:ALK4 heteromultimers comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 amino acids 29-109 of SEQ ID NO: 2. In some embodiments, ActRIIB:ALK4 heteromultimers comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 amino acids 25-131 of SEQ ID NO: 2. In certain embodiments, ActRIIB:ALK4 heteromultimer complexes of the disclosure comprise at least one ActRIIB polypeptide wherein the position corresponding to L79 of SEQ ID NO: 2 is not an acidic amino acid (i.e., not naturally occurring D or E amino acid residues or an artificial acidic amino acid residue). ActRIIB:ALK4 heteromultimers of the disclosure include, e.g., heterodimers, heterotrimers, heterotetramers and further higher order oligomeric structures. See, e.g., FIGS. 11-13, which may also be applied to ActRII:ALK7 oligomeric structures. In certain preferred embodiments, heteromultimer complexes of the disclosure are ActRIIB:ALK7 heterodimers.

In certain embodiments, the disclosure relates to a heteromultimer comprising at least one ALK7-Fc fusion polypeptide and at least one ActRIIB-Fc fusion polypeptide. In some embodiments, an ActRIIB-Fc:ALK7-Fc heteromultimers binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, an ActRIIB-Fc:ALK7-Fc heteromultimers inhibit signaling of one or more ActRII-ALK4 (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, an ActRIIB-Fc:ALK7-Fc heteromultimers is a heterodimer.

In certain embodiments, the disclosure relates to heteromultimers that comprise at least one ALK7 polypeptide, which includes fragments, functional variants, and modified forms thereof. Preferably, ALK7 polypeptides for use as disclosed herein (e.g., heteromultimers comprising an ALK7 polypeptide and uses thereof) are soluble (e.g., an extracellular domain of ALK7). In other preferred embodiments, ALK7 polypeptides for use as disclosed herein bind to and/or inhibit (antagonize) activity (e.g., induction of Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) superfamily ligands. In some embodiments, the ALK7-Fc fusion polypeptide comprises an ALK7 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 an amino acid sequence that begins at any one of amino acids 21-28 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, and 28) SEQ ID NO: 120, 121, or 122, and ends at any one of amino acids 92-113 (e.g., amino acid residues 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, and 113) of SEQ ID NO: 120, 121, or 122. In some embodiments, the ALK7-Fc fusion polypeptide comprises an ALK7 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 28-92 of SEQ ID NOs: 120, 121, or 122. In some embodiments, the ALK7-Fc fusion polypeptide comprises an ALK7 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 21-113 of SEQ ID NOs: 120, 121, or 122. In some embodiments, the ALK7-Fc fusion polypeptide comprises an ALK7 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: 120, 123, 124, 125, 121, 126, 122, 127, 128, 129, 130, 131, 132, 133, or 134. In some embodiments, heteromultimers of the disclosure consist or consist essentially of at least one ALK7 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 NO: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, or 134.

In certain aspects, the present disclosure relates to heteromultimer complexes comprising one or more ALK7 receptor polypeptides (e.g., SEQ ID Nos: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, 134 and variants thereof) and one or more ActRIIB receptor polypeptides (e.g., 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, 409 and variants thereof), which are generally referred to herein as “ActRIIB:ALK7 heteromultimer” or “ActRIIB-ALK7 heteromultimers”, including uses thereof (e.g., treating heart failure in a patient in need thereof). Preferably, ActRIIB-ALK7 heteromultimers are soluble [e.g., a heteromultimer complex comprises a soluble portion (domain) of an ALK7 receptor and a soluble portion (domain) of an ActRIIB receptor]. In general, the extracellular domains of ALK7 and ActRIIB correspond to soluble portion of these receptors. Therefore, in some embodiments, ActRIIB-ALK7 heteromultimers comprise an extracellular domain of an ALK7 receptor and an extracellular domain of an ActRIIB receptor. In some embodiments, ActRIIB-ALK7 heteromultimers inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIB-ALK7 heteromultimers bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIB-ALK7 heteromultimers comprise at least one ALK7 polypeptide that comprises, consists essentially of, or consists of a 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: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, and 134. In some embodiments, ActRIIB-ALK7 heteromultimers comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 any one of SEQ ID NOs: 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, ActRIIB-ALK7 heteromultimer complexes of the disclosure comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 a portion of ActRIIB beginning at a residue corresponding to any one of amino acids 20-29, 20-24, 21-24, 22-25, or 21-29 and end at a position from 109-134, 119-134, 119-133, 129-134, or 129-133 of SEQ ID NO: 2. In some embodiments, ActRIIB-ALK7 heteromultimers comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 amino acids 29-109 of SEQ ID NO: 2. In some embodiments, ActRIIB-ALK7 heteromultimers comprise at least one ActRIIB polypeptide that comprises, consists essentially of, consists of a 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 amino acids 25-131 of SEQ ID NO: 2. In certain embodiments, ActRIIB-ALK7 heteromultimer complexes of the disclosure comprise at least one ActRIIB polypeptide wherein the position corresponding to L79 of SEQ ID NO: 2 is not an acidic amino acid (i.e., not naturally occurring D or E amino acid residues or an artificial acidic amino acid residue). ActRIIB-ALK7 heteromultimers of the disclosure include, e.g., heterodimers, heterotrimers, heterotetramers and further higher order oligomeric structures. See, e.g., FIGS. 11-13, which may also be applied to both ActRII-ALK4 and ActRII-ALK7 oligomeric structures. In certain preferred embodiments, heteromultimer complexes of the disclosure are ActRIIB-ALK7 heterodimers.

In certain aspects, the present disclosure relates to heteromultimer complexes comprising one or more ALK7 receptor polypeptides (e.g., SEQ ID Nos: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, 134 and variants thereof) and one or more ActRIIA receptor polypeptides (e.g., SEQ ID NOs: 364, 366, 367, 368, 369, 378, 380, 381, 384 and variants thereof), which are generally referred to herein as “ActRIIA:ALK7 heteromultimer” or “ActRIIA-ALK7 heteromultimers”, including uses thereof (e.g., treating heart failure in a patient in need thereof). Preferably, ActRIIA-ALK7 heteromultimers are soluble [e.g., a heteromultimer complex comprises a soluble portion (domain) of an ALK7 receptor and a soluble portion (domain) of an ActRIIA receptor]. In general, the extracellular domains of ALK7 and ActRIIA correspond to soluble portion of these receptors. Therefore, in some embodiments, ActRIIA-ALK7 heteromultimers comprise an extracellular domain of an ALK7 receptor and an extracellular domain of an ActRIIA receptor. In some embodiments, ActRIIA-ALK7 heteromultimers inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIA-ALK7 heteromultimers bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIA-ALK7 heteromultimers comprise at least one ALK7 polypeptide that comprises, consists essentially of, or consists of a 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: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, and 134. In some embodiments, ActRIIA-ALK7 heteromultimers comprise at least one ActRIIA polypeptide that comprises, consists essentially of, consists of a 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 any one of SEQ ID NOs: 364, 366, 367, 368, 369, 378, 380, 381, 384. In certain preferred embodiments, heteromultimer complexes of the disclosure are ActRIIA-ALK7 heterodimers.

In certain aspects, the present disclosure relates to heteromultimer complexes comprising one or more ALK4 receptor polypeptides (e.g., SEQ ID Nos: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, 422 and variants thereof) and one or more ActRIIA receptor polypeptides (e.g., SEQ ID NOs: 364, 366, 367, 368, 369, 378, 380, 381, 384 and variants thereof), which are generally referred to herein as “ActRIIA:ALK4 heteromultimer” or “ActRIIA-ALK4 heteromultimers”, including uses thereof (e.g., treating heart failure in a patient in need thereof). Preferably, ActRIIA-ALK4 heteromultimers are soluble [e.g., a heteromultimer complex comprises a soluble portion (domain) of an ALK4 receptor and a soluble portion (domain) of an ActRIIA receptor]. In general, the extracellular domains of ALK4 and ActRIIA correspond to soluble portion of these receptors. Therefore, in some embodiments, ActRIIA-ALK4 heteromultimers comprise an extracellular domain of an ALK4 receptor and an extracellular domain of an ActRIIA receptor. In some embodiments, ActRIIA-ALK4 heteromultimers inhibit (e.g., Smad signaling) of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIA-ALK4 heteromultimers bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, ActRIIA-ALK4 heteromultimers comprise at least one ALK4 polypeptide that comprises, consists essentially of, or consists of a 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: 84, 85, 86, 87, 88, 89, 92, 93, 247, 249, 421, and 422. In some embodiments, ActRIIA-ALK4 heteromultimer complexes of the disclosure comprise at least one ALK4 polypeptide that comprises, consists essentially of, consists of a 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 a portion of ALK4 beginning at a residue corresponding to any one of amino acids 24-34, 25-34, or 26-34 of SEQ ID NO: 84 and ending at a position from 101-126, 102-126, 101-125, 101-124, 101-121, 111-126, 111-125, 111-124, 121-126, 121-125, 121-124, or 124-126 of SEQ ID NO: 84. In some embodiments, ActRIIA-ALK4 heteromultimers comprise at least one ALK4 polypeptide that comprises, consists essentially of, consists of a 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 amino acids 34-101 with respect to SEQ ID NO: 84. In some embodiments, ActRIIA-ALK4 heteromultimers comprise at least one ActRIIA polypeptide that comprises, consists essentially of, consists of a 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 any one of SEQ ID NOs: 364, 366, 367, 368, 369, 378, 380, 381, 384. In certain preferred embodiments, heteromultimer complexes of the disclosure are ActRIIA-ALK4 heterodimers.

In certain embodiments, the disclosure relates to a heteromultimer comprising a first ActRIIA-Fc fusion polypeptide and a second ActRIIA-Fc fusion polypeptide, wherein the second variant ActRIIA-Fc fusion polypeptide differs from that present in the first polypeptide. In some embodiments, an ActRIIA-Fc:ActRIIA-Fc heteromultimers binds to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, an ActRIIA-Fc:ActRIIA-Fc heteromultimers inhibit signaling of one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10). In some embodiments, an ActRIIA-Fc:ActRIIA-Fc heteromultimers is a heterodimer.

II. Linkers

The disclosure provides for an ActRII-ALK4 ligand trap polypeptide (e.g., ActRIIB, ActRIIA, ALK4, ALK7, and follistatin polypeptides including variants thereof) that may be fused to an additional polypeptide disclosed herein including, for example, fused to a heterologous portion (e.g., an Fc portion). In these embodiments, the polypeptide portion (e.g., ActRIIB, ActRIIA, ALK4, ALK7, and follistatin polypeptides including variants thereof) is connected to the additional polypeptide (e.g., a heterologous portion such as an Fc domain) by means of a linker. In some embodiments, the linkers are glycine and serine rich linkers. In some embodiments, the linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or glycine and proline residues and may, for example, contain a single sequence of threonine/serine and glycines or repeating sequences of threonine/serine and/or glycines, e.g., GGG (SEQ ID NO: 261), GGGG (SEQ ID NO: 262), TGGGG (SEQ ID NO: 263), SGGGG (SEQ ID NO: 264), TGGG (SEQ ID NO: 265), or SGGG (SEQ ID NO: 266) singlets, or repeats. Other near neutral amino acids, such as, but not limited to, Thr, Asn, Pro and Ala, may also be used in the linker sequence. In some embodiments, the linker comprises various permutations of amino acid sequences containing Gly and Ser. In some embodiments, the linker is greater than 10 amino acids in length. In further embodiments, the linkers have a length of at least 12, 15, 20, 21, 25, 30, 35, 40, 45 or 50 amino acids. In some embodiments, the linker is less than 40, 35, 30, 25, 22 or 20 amino acids. In some embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-21, 10-15, 10, 15-25, 17-22, 20, or 21 amino acids in length. In preferred embodiments, the linker comprises the amino acid sequence GlyGlyGlyGlySer (GGGGS) (SEQ ID NO: 267), or repetitions thereof (GGGGS)n, where n≥2. In particular embodiments n≥3, or n=3-10. In some embodiments, n≥4, or n=4-10. In some embodiments, n is not greater than 4 in a (GGGGS)n linker. In some embodiments,n=4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6. In some embodiments, n=3, 4, 5, 6, or 7. In particular embodiments, n=4. In some embodiments, a linker comprising a (GGGGS)n sequence also comprises an N-terminal threonine. In some embodiments, the linker is any one of the following:

(SEQ ID NO: 268)
GGGGSGGGGS
(SEQ ID NO: 269)
TGGGGSGGGGS
(SEQ ID NO: 270)
TGGGGSGGGGSGGGGS
(SEQ ID NO: 271)
TGGGGSGGGGSGGGGSGGGGS
(SEQ ID NO: 272)
TGGGGSGGGGSGGGGSGGGGSGGGGS
(SEQ ID NO: 273)
TGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
or
(SEQ ID NO: 274)
TGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

In some embodiments, the linker comprises the amino acid sequence of TGGGPKSCDK (SEQ ID NO: 275). In some embodiments, the linker is any one of SEQ ID NOs: 268-275 lacking the N-terminal threonine. In some embodiments, the linker does not comprise the amino acid sequence of SEQ ID NO: 273 or 274.

In some embodiments, a polypeptide described (e.g., ActRIIB, ActRIIA, ALK4, ALK7, and follistatin, polypeptides including variants thereof) herein may include a polypeptide fused to a moiety by way of a linker. In some embodiments, the moiety increases stability of the polypeptide. In some embodiments, the moiety is selected from the group consisting of an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or a human serum albumin. Suitable peptide linkers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, a linker can contain motifs, e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG (SEQ ID NO: 261), GGGA (SEQ ID NO: 280), GGGS (SEQ ID NO: 281), GGGG (SEQ ID NO: 262), GGGGA (SEQ ID NO: 282), GGGGS (SEQ ID NO: 267), GGGGG (SEQ ID NO: 283), GGAG (SEQ ID NO: 284), GGSG (SEQ ID NO: 285), AGGG (SEQ ID NO: 286), or SGGG (SEQ ID NO: 266). In some embodiments, a linker can contain 2 to 12 amino acids including motifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 287), GSGS (SEQ ID NO: 288), GAGAGA (SEQ ID NO: 289), GSGSGS (SEQ ID NO: 290), GAGAGAGA (SEQ ID NO: 291), GSGSGSGS (SEQ ID NO: 292), GAGAGAGAGA (SEQ ID NO: 293), GSGSGSGSGS (SEQ ID NO: 294), GAGAGAGAGAGA (SEQ ID NO: 295), and GSGSGSGSGSGS (SEQ ID NO: 296). In some embodiments, a linker can contain 3 to 12 amino acids including motifs of GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 297), GGSGGS (SEQ ID NO: 298), GGAGGAGGA (SEQ ID NO: 299), GGSGGSGGS (SEQ ID NO: 300), GGAGGAGGAGGA (SEQ ID NO: 301), and GGSGGSGGSGGS (SEQ ID NO: 302). In some embodiments, a linker can contain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 303), GGSG (SEQ ID NO: 304), GGAGGGAG (SEQ ID NO: 305), GGSGGGSG (SEQ ID NO: 306), GGAGGGAGGGAG (SEQ ID NO: 307), and GGSGGGSGGGSG (SEQ ID NO: 308). In some embodiments, a linker can contain motifs of GGGGA (SEQ ID NO: 309) or GGGGS (SEQ ID NO: 267), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 310) and GGGGSGGGGSGGGGS (SEQ ID NO: 311). In some embodiments, an amino acid linker between a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or a human serum albumin) and a polypeptide (e.g., ActRIIB, ActRIIA, ALK4, ALK7, and follistatin polypeptides including variants thereof) may be GGG, GGGA (SEQ ID NO: 280), GGGG (SEQ ID NO: 262), GGGAG (SEQ ID NO: 312), GGGAGG (SEQ ID NO: 313), or GGGAGGG (SEQ ID NO: 314).

In some embodiments, a linker can also contain amino acids other than glycine, alanine, and serine, e.g., AAAL (SEQ ID NO: 315), AAAK (SEQ ID NO: 316), AAAR (SEQ ID NO: 317), EGKSSGSGSESKST (SEQ ID NO: 318), GSAGSAAGSGEF (SEQ ID NO: 319), AEAAAKEAAAKA (SEQ ID NO: 320), KESGSVSSEQLAQFRSLD (SEQ ID NO: 321), GENLYFQSGG (SEQ ID NO: 322), SACYCELS (SEQ ID NO: 323), RSIAT (SEQ ID NO: 324), RPACKIPNDLKQKVMNH (SEQ ID NO: 325), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 326), AAANSSIDLISVPVDSR (SEQ ID NO: 327), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 328). In some embodiments, a linker can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO: 329). In some embodiments, a linker can contain motifs, e.g., multiple or repeating motifs, of praline-rich sequences such as (XP)n, in which X may be any amino acid (e.g., A, K, or E) and n is from 1-5, and PAPAP (SEQ ID NO: 330).

The length of the peptide linker and the amino acids used can be adjusted depending on the two polypeptides involved and the degree of flexibility desired in the final polypeptide fusion polypeptide. The length of the linker can be adjusted to ensure proper polypeptide folding and avoid aggregate formation.

H) Polypeptide Variants and Modifications

In part, the disclosure relates to ActRII-ALK4 antagonists that are variant polypeptides (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). Variant polypeptides of the disclosure included, for example, variant polypeptides produced by one or more amino acid substitutions, deletions, additions or combinations thereof as well as variants of one or more post-translational modifications (e.g., including, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation). Methods for generating variant polypeptides comprising one or more amino acid modifications, particularly methods for generating variant polypeptides that have one or more desired properties, are described herein or otherwise well known in the art. Likewise, various methods for determining if a variant polypeptide has retained or developed one or more desired properties (e.g., alterations in ligand binding and/or antagonistic activities) are described herein or otherwise well known in the art. These methods can be used to generate variant polypeptides (e.g., variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptides) as well as validate their activity (or other properties) as described here.

As described above, the disclosure provides polypeptides (e.g., ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptides) sharing a specified degree of sequence identity or similarity to a naturally occurring polypeptide. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid “identity” is equivalent to amino acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com). In a specific embodiment, the following parameters are used in the GAP program: either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com). Exemplary parameters include using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified, percent identity between two amino acid sequences is to be determined using the GAP program using a Blosum 62 matrix, a GAP weight of 10 and a length weight of 3, and if such algorithm cannot compute the desired percent identity, a suitable alternative disclosed herein should be selected.

In another embodiment, the percent identity between two amino acid sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Another embodiment for determining the best overall alignment between two amino acid sequences can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity. In one embodiment, amino acid sequence identity is performed using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)). In a specific embodiment, parameters employed to calculate percent identity and similarity of an amino acid alignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5 and Gap Size Penalty=0.05.

In some embodiments, the disclosure contemplates making functional variant polypeptides by modifying the structure of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) for such purposes as enhancing therapeutic efficacy or stability (e.g., shelf-life and resistance to proteolytic degradation in vivo). Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a polypeptide of the disclosure results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide, or to bind to one or more ActRII-ALK4 ligands including, for example, activin A, activin B, GDF8, GDF11, BMP6, and BMP10.

In certain embodiments, the disclosure contemplates specific mutations of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) so as to alter the glycosylation of the polypeptide. Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where “X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the polypeptide (for O-linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties present on a polypeptide may be accomplished chemically and/or enzymatically. Chemical deglycosylation may involve, for example, exposure of a polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987) 138:350]. The sequence of a polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect, and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, polypeptides of the present disclosure for use in humans may be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well. In some embodiments, polypeptides of the disclosure (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptides) are glycosylated and have a glycosylation pattern obtainable from of the polypeptide in a CHO cell.

The disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) as well as truncation mutants. Pools of combinatorial mutants are especially useful for identifying functionally active (e.g., ActRII-ALK4 ligand binding) sequences. The purpose of screening such combinatorial libraries may be to generate, for example, polypeptides variants which have altered properties, such as altered pharmacokinetic or altered ligand binding. A variety of screening assays are provided below, and such assays may be used to evaluate variants. For example, polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) variants, homomultimers, and heteromultimers comprising the same, may be screened for ability to bind to one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), to prevent binding of an ActRII-ALK4 ligand to an ActRII and/or ALK4 polypeptide, as well as homomultimers of heteromultimers thereof, and/or to interfere with signaling caused by an ActRII-ALK4 ligand.

The activity of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof, or variant thereof may also be tested in a cell-based or in vivo assay. For example, the effect of a polypeptide, including homomultimers and heteromultimers thereof, or a variant thereof on the expression of genes involved in heart failure pathogenesis assessed. This may, as needed, be performed in the presence of one or more recombinant ligand proteins (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), and cells may be transfected so as to produce polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) r, and optionally, an ActRII-ALK4 ligand. Likewise, a polypeptide, including homomultimers and heteromultimers thereof, or a variant thereof may be administered to a mouse or other animal and effects on heart failure pathogenesis may be assessed using art-recognized methods. Similarly, the activity of a polypeptide, including homomultimers and heteromultimers thereof, or variant thereof may be tested in blood cell precursor cells for any effect on growth of these cells, for example, by the assays as described herein and those of common knowledge in the art. A SMAD-responsive reporter gene may be used in such cell lines to monitor effects on downstream signaling.

In certain aspects, a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including heteromultimers or homomultimers thereof, of the disclosure bind to one or more ActRII-ALK4 ligands. In some embodiments, a polypeptide, including heteromultimers or homomultimers thereof, of the disclosure bind to one or more ActRII-ALK4 ligands with a K_D of at least 1×10⁻⁷ M. In some embodiments, the one or more ActRII-ALK4 ligands is selected from the group consisting of: activin A, activin B, GDF8, GDF11, and BMP10.

In certain aspects, a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including heteromultimers or homomultimers thereof, of the disclosure inhibits one or more ActRII-ALK4 family ligands. In some embodiments, a polypeptide, including heteromultimers or homomultimers thereof, of the disclosure inhibits signaling of one or more ActRII-ALK4 ligands. In some embodiments, a polypeptide, including heteromultimers or homomultimers thereof, of the disclosure inhibits Smad signaling of one or more ActRII-ALK4 ligands. In some embodiments, a polypeptide, including heteromultimers or homomultimers thereof, of the disclosure inhibits signaling of one or more ActRII-ALK4 ligands in a cell-based assay. In some embodiments, a polypeptide, including heteromultimers or homomultimers thereof, of the disclosure inhibits one or more ActRII-ALK4 ligands selected from the group consisting of activin A, activin B, GDF8, GDF11, and BMP10.

Combinatorial-derived variants can be generated which have increased selectivity or generally increased potency relative to a reference polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof. Such variants, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding unmodified a polypeptide, including homomultimers and heteromultimers thereof. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction, or otherwise inactivation, of an unmodified polypeptide. Such variants, and the genes which encode them, can be utilized to alter polypeptide complex levels by modulating the half-life of the polypeptide. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant polypeptide complex levels within the cell. In an Fc fusion protein, mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the polypeptide, including homomultimers and heteromultimers thereof.

A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ActRIIA, ActRIIB, ALK4, ALK7, or follistatin encoding nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).

There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art [Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; and Ike et al. (1983) Nucleic Acid Res. 11:477]. Such techniques have been employed in the directed evolution of other proteins [Scott et al., (1990) Science 249:386-390; Roberts et al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815].

Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof of the disclosure can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085], by linker scanning mutagenesis [Gustin et al. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982) Science 232:316], by saturation mutagenesis [Meyers et al., (1986) Science 232:613]; by PCR mutagenesis [Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by random mutagenesis, including chemical mutagenesis [Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol Biol 7:32-34]. Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated (bioactive) forms of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof. The most widely used techniques for screening large gene libraries typically comprise cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Preferred assays include ligand (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) binding assays and/or ligand-mediated cell signaling assays.

As will be recognized by one of skill in the art, most of the described mutations, variants or modifications described herein may be made at the nucleic acid level or, in some cases, by post-translational modification or chemical synthesis. Such techniques are well known in the art and some of which are described herein. In part, the present disclosure identifies functionally active portions (fragments) and variants of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof that can be used as guidance for generating and using other variant polypeptides within the scope of the methods and uses described herein.

In certain embodiments, functionally active fragments of a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof of the disclosure can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding polypeptides disclosed herein. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ActRII and/or ALK4 receptors and/or one or more ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10).

In certain embodiments, a polypeptide (e.g., an ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), including homomultimers and heteromultimers thereof or variant thereof of the disclosure may further comprise post-translational modifications in addition to any that are naturally present in the polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the polypeptide, including homomultimers and heteromultimers thereof, may contain non-amino acid elements, such as polyethylene glycols, lipids, polysaccharide or monosaccharide, and phosphates. Effects of such non-amino acid elements on the functionality of a polypeptide may be tested as described herein for other polypeptide variants. When a polypeptide of the disclosure is produced in cells by cleaving a nascent form of the polypeptide, post-translational processing may also be important for correct folding and/or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the polypeptides.

I) Nucleic Acids and Method of Manufacture

In certain aspects, the disclosure provides isolated and/or recombinant nucleic acids encoding any of the polypeptides disclosed herein including, for example, ActRIIB, ActRIIA, ALK4, or ALK7 polypeptides (e.g., soluble ActRIIB, ActRIIA, ALK4, or ALK7 polypeptides), or follistatin polypeptides, as well as any of the variants disclosed herein. For example, SEQ ID NO: 4 encodes a naturally occurring ActRIIB precursor polypeptide, while SEQ ID NO: 3 encodes a soluble ActRIIB polypeptide. The subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids are may be used, for example, in methods for making ActRIIB, ActRIIA, ALK4, or ALK7 polypeptides or as direct therapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the disclosure relates to isolated and/or recombinant nucleic acids comprising a coding sequence for one or more of the ActRIIB, ActRIIA, ALK4, ALK7, or follistatin polypeptide(s) as described herein. For example, in some embodiments, the disclosure relates to an isolated and/or recombinant nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424. In some embodiments, an isolated and/or recombinant polynucleotide sequence of the disclosure comprises a promoter sequence operably linked to a coding sequence described herein (e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424). In some embodiments, the disclosure relates to vectors comprising an isolated and/or recombinant nucleic acid described herein (e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424). In some embodiments, the disclosure relates to a cell comprising an isolated and/or recombinant polynucleotide sequence described herein (e.g., a nucleic acid that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence corresponding to any one of SEQ ID Nos: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424). In some embodiments, the cell is a CHO cell. In some embodiments, the cell is a COS cell.

In certain embodiments, nucleic acids encoding variant ActRIIB (or homomultimers or heteromultimers thereof), ALK4 or ALK7 polypeptides of the disclosure are understood to include nucleic acids that are variants of any one of SEQ ID NOs: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions including allelic variants, and therefore, will include coding sequence that differ from the nucleotide sequence designated in any one of SEQ ID NOs: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424.

In certain embodiments, variant ActRIIB (or homomultimers or heteromultimers thereof), ALK4, or ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 3, 4, 10, 32, 35, 38, 41, 44, 47, 221, 222, 223, 224, 233, 234, 235, 236, 237, 238, 239, 240, 243, 248, 250, 251, 252, 255, 277, 331, 334, 337, 340, 343, 346, 349, 352, 355, 369, 370, 382, 397, 407, 423, and 424. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47.

In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 277. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 331. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 334. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 337. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 340. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 343. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 346. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 349. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 352. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 355. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 382. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 397. In certain embodiments, variant ActRIIB polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 407.

In certain embodiments, variant ActRIIA polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 369. In certain embodiments, variant ActRIIA polypeptides (or homomultimers or heteromultimers thereof) of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 370.

In certain embodiments, ALK4 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 221. In certain embodiments, ALK4 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 222. In certain embodiments, ALK4 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 223. In certain embodiments, ALK4 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 224. In certain embodiments, ALK4 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 423. In certain embodiments, ALK4 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 424.

In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 233. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 234. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 235. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 236. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 237. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 238. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 239. In certain embodiments, ALK7 polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 240.

In certain embodiments, ALK4-Fc fusion polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 243. In certain embodiments, ALK4-Fc fusion polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 248. In certain embodiments, ALK4-Fc fusion polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 250. In certain embodiments, ALK4-Fc fusion polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 251. In certain embodiments, ALK4-Fc fusion polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 252. In certain embodiments, ALK7-Fc fusion polypeptides of the disclosure are encoded by isolated and/or recombinant nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 255.

In certain aspects, the subject nucleic acids encoding variant ActRIIB polypeptides are further understood to include nucleic acids that are variants of SEQ ID NO: 3. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include coding sequences that differ from the nucleotide sequence of the coding sequence designated in SEQ ID NO: 4.

In certain embodiments, the disclosure provides isolated or recombinant nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NO: 3, and variants of SEQ ID NO: 3 are also within the scope of this disclosure. In further embodiments, the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also include nucleotide sequences that hybridize under highly stringent conditions to nucleic acids encoding ActRIIB or ActRIIA polypeptides in either homomeric or heteromeric forms, ALK4, or ALK7 polypeptides of the disclosure, or follistatin polypeptides of the disclosure, the complement sequence, or fragments thereof. As discussed above, one of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. One of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, the disclosure provides nucleic acids which hybridize under low stringency conditions of 6×SSC at room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forth in the disclosure due to degeneracy in the genetic code are also within the scope of the disclosure. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the polypeptide. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject polypeptides will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular polypeptide may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

In certain aspects, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). Such useful expression control sequences, include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of polypeptide desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other polypeptide encoded by the vector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid of the disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant variant ActRIIB polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of polypeptides in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production of the polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif) and pCI-neo vectors (Promega, Madison, Wisc.). As will be apparent, the subject gene constructs can be used to cause expression of the polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) in cells propagated in culture, e.g., to produce polypeptides, including fusion polypeptides or polypeptides, for purification.

In certain embodiments, the disclosure relates to methods of making polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) as well as homomultimer and heteromultimers comprising the same, as described herein. Such a method may include expressing any of the nucleic acids disclosed herein in a suitable cell (e.g., a CHO cell or COS cell). Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), wherein said cell comprise with an expression construct of polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). In some embodiments, the method further comprises recovering the expressed polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). Polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) may be recovered as crude, partially purified or highly purified fractions using any of the well-known techniques for obtaining protein from cell cultures.

This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence for one or more polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). The host cell may be any prokaryotic or eukaryotic cell. For example, polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods of producing polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). For example, a host cell transfected with an expression vector encoding polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) can be cultured under appropriate conditions to allow expression of the polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) to occur. The polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) may be secreted and isolated from a mixture of cells and medium containing the polypeptides. Alternatively, the polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The subject polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying polypeptides, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide). In a preferred embodiment, the polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) are fusion polypeptides containing a domain which facilitates purification.

In preferred embodiments, ActRII polypeptides, ALK4 polypeptides, ALK7 polypeptides, and ActRIIB-ALK4, ActRIIB-ALK7, ActRIIA-ALK4, and ActRIIA-ALK7 heteromultimers to be used in accordance with the methods described herein are isolated polypeptides. As used herein, an isolated protein or polypeptide is one which has been separated from a component of its natural environment. In some embodiments, a polypeptide of the disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). Methods for assessment of purity are well known in the art [see, e.g., Flatman et al., (2007) J. Chromatogr. B 848:79-87]. In some embodiments, ActRII polypeptides, ALK4 polypeptides, and ActRIIB-ALK4 heteromultimers to be used in accordance with the methods described herein are recombinant polypeptides.

In certain embodiments, ActRIIB or ActRIIA polypeptides of the disclosure can be produced by a variety of art-known techniques. For example, such ActRIIB or ActRIIA polypeptides can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively, the ActRIIB or ActRIIA polypeptides, fragments or variants thereof may be recombinantly produced using various expression systems (e.g., E. coli, Chinese Hamster Ovary cells, COS cells, baculovirus) as is well known in the art (also see above). In a further embodiment, the ActRIIB or ActRIIA polypeptides may be produced by digestion of naturally occurring or recombinantly produced full-length ActRIIB or ActRIIA polypeptides by using, for example, a protease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using a commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such ActRIIB or ActRIIA polypeptides may be produced from naturally occurring or recombinantly produced full-length ActRIIB or ActRIIA polypeptides such as standard techniques known in the art, such as by chemical cleavage (e.g., cyanogen bromide, hydroxylamine).

In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide), can allow purification of the expressed fusion polypeptide by affinity chromatography using a Ni²⁺ metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified polypeptides of the disclosure (e.g., a variant ActRIIA, ActRIIB, ALK4, ALK7, or follistatin polypeptide) (e.g., see Hochuli et al., (1987) J Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

3. Antibody Antagonists

In certain aspects, an ActRII-ALK4 antagonist to be 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 (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure) is an antibody (ActRII-ALK4 antagonist antibody), or combination of antibodies. An ActRII-ALK4 antagonist antibody, or combination of antibodies, may bind to, for example, one or more ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). As described herein, ActRII-ALK4 antagonist antibodies may be used, alone or in combination with one or more supportive therapies or active agents, to treat, prevent, or reduce the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies), particularly treating, preventing or reducing the progression rate and/or severity of one or more heart failure-associated complications.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE, and/or activin BE). Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least activin. As used herein, an activin antibody (or anti-activin antibody) generally refers to an antibody that binds to activin with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting activin. In certain embodiments, the extent of binding of an activin antibody to an unrelated, non-activin protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to activin as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein interaction or binding affinity assay. In certain embodiments, an activin antibody binds to an epitope of activin that is conserved among activin from different species. In certain preferred embodiments, an anti-activin antibody binds to human activin. In some embodiments, an activin antibody may inhibit activin from binding to a type I and/or type II receptor (e.g., ActRIIA, ActRIIB, and/or ALK4) and thus inhibit activin-mediated signaling (e.g., Smad signaling). It should be noted that activin A has similar sequence homology to activin B and therefore antibodies that bind to activin A, in some instances, may also bind to and/or inhibit activin B, which also applies to anti-activin B antibodies. In some embodiments, the disclosure relates to a multispecific antibody (e.g., bi-specific antibody), and uses thereof, that binds to activin and further binds to, for example, one or more additional ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), one or more type I receptor and/or type II receptors (e.g., ActRIIA, ActRIIB, and/or ALK4). In some embodiments, a multispecific antibody that binds to activin does not bind or does not substantially bind to BMP9 (e.g., binds to BMP9 with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecific antibody that binds to activin does not bind or does not substantially bind to activin A (e.g., binds to activin A with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises an activin antibody and one or more additional antibodies that bind to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a combination of antibodies that comprises an activin antibody does not comprise a BMP9 antibody.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least activin B. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least activin B. As used herein, an activin B antibody (or anti-activin B antibody) generally refers to an antibody that binds to activin B with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting activin B. In certain embodiments, the extent of binding of an activin B antibody to an unrelated, non-activin B protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to activin as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein interaction or binding affinity assay. In certain embodiments, an activin B antibody binds to an epitope of activin B that is conserved among activin B from different species. In certain preferred embodiments, an anti-activin B antibody binds to human activin B. In some embodiments, an activin B antibody may inhibit activin B from binding to a type I and/or type II receptor (e.g., ActRIIA, ActRIIB, and/or ALK4) and thus inhibit activin B-mediated signaling (e.g., Smad signaling). In some embodiments, an activin B antibody may inhibit activin B from binding to a co-receptor and thus inhibit activin B-mediated signaling (e.g., Smad signaling). It should be noted that activin B has similar sequence homology to activin A and therefore antibodies that bind to activin B, in some instances, may also bind to and/or inhibit activin A. In some embodiments, the disclosure relates to a multispecific antibody (e.g., bi-specific antibody), and uses thereof, that binds to activin B and further binds to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a multispecific antibody that binds to activin B does not bind or does not substantially bind to BMP9 (e.g., binds to BMP9 with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecific antibody that binds to activin B does not bind or does not substantially bind to activin A (e.g., binds to activin A with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises an activin B antibody and one or more additional antibodies that bind to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a combination of antibodies that comprises an activin B antibody does not comprise a BMP9 antibody. In some embodiments, a combination of antibodies that comprises an activin B antibody does not comprise an activin A antibody.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least GDF8. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least GDF8. As used herein, a GDF8 antibody (or anti-GDF8 antibody) generally refers to an antibody that binds to GDF8 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF8. In certain embodiments, the extent of binding of a GDF8 antibody to an unrelated, non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to GDF8 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein interaction or binding affinity assay. In certain embodiments, a GDF8 antibody binds to an epitope of GDF8 that is conserved among GDF8 from different species. In certain preferred embodiments, an anti-GDF8 antibody binds to human GDF8. In some embodiments, a GDF8 antibody may inhibit GDF8 from binding to a type I and/or type II receptor (e.g., ActRIIA, ActRIIB, and/or ALK4) and thus inhibit GDF8-mediated signaling (e.g., Smad signaling). In some embodiments, a GDF8 antibody may inhibit GDF8 from binding to a co-receptor and thus inhibit GDF8-mediated signaling (e.g., Smad signaling). It should be noted that GDF8 has high sequence homology to GDF11 and therefore antibodies that bind to GDF8, in some instances, may also bind to and/or inhibit GDF11. In some embodiments, the disclosure relates to a multispecific antibody (e.g., bi-specific antibody), and uses thereof, that binds to GDF8 and further binds to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a multispecific antibody that binds to GDF8 does not bind or does not substantially bind to BMP9 (e.g., binds to BMP9 with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecific antibody that binds to GDF8 does not bind or does not substantially bind to activin A (e.g., binds to activin A with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises a GDF8 antibody and one or more additional antibodies that bind to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a combination of antibodies that comprises a GDF8 antibody does not comprise a BMP9 antibody. In some embodiments, a combination of antibodies that comprises a GDF8 antibody does not comprise an activin A antibody.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least GDF11. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least GDF11. As used herein, a GDF11 antibody (or anti-GDFT11 antibody) generally refers to an antibody that binds to GDF11 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF11. In certain embodiments, the extent of binding of a GDF11 antibody to an unrelated, non-GDF11 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to GDF11 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein interaction or binding affinity assay. In certain embodiments, a GDF11 antibody binds to an epitope of GDF11 that is conserved among GDF11 from different species. In certain preferred embodiments, an anti-GDF11 antibody binds to human GDF11. In some embodiments, a GDF11 antibody may inhibit GDF11 from binding to a type I and/or type II receptor (e.g., ActRIIA, ActRIIB, and/or ALK4) and thus inhibit GDF11-mediated signaling (e.g., Smad signaling). In some embodiments, a GDF11 antibody may inhibit GDF11 from binding to a co-receptor and thus inhibit GDF11-mediated signaling (e.g., Smad signaling). It should be noted that GDF11 has high sequence homology to GDF8 and therefore antibodies that bind to GDF11, in some instances, may also bind to and/or inhibit GDF8. In some embodiments, the disclosure relates to a multispecific antibody (e.g., bi-specific antibody), and uses thereof, that binds to GDF11 and further binds to, for example, one or more additional ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), one or more type I receptor and/or type II receptors (e.g., ActRIIA, ActRIIB, and/or ALK4), and/or one or more co-receptors. In some embodiments, a multispecific antibody that binds to GDF11 does not bind or does not substantially bind to BMP9 (e.g., binds to BMP9 with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecific antibody that binds to GDF11 does not bind or does not substantially bind to activin A (e.g., binds to activin A with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises a GDF11 antibody and one or more additional antibodies that bind to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a combination of antibodies that comprises a GDF11 antibody does not comprise a BMP9 antibody. In some embodiments, a combination of antibodies that comprises a GDF11 antibody does not comprise an activin A antibody.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least BMP6. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least BMP6. As used herein, a BMP6 antibody (or anti-BMP6 antibody) generally refers to an antibody that can bind to BMP6 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting BMP6. In certain embodiments, the extent of binding of a BMP6 antibody to an unrelated, non-BMP6 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to BMP6 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein interaction or binding affinity assay. In certain embodiments, a BMP6 antibody binds to an epitope of BMP6 that is conserved among BMP6 from different species. In certain preferred embodiments, an anti-BMP6 antibody binds to human BMP6. In some embodiments, a BMP6 antibody may inhibit BMP6 from binding to a type I and/or type II receptor (e.g., ActRIIA, ActRIIB, and/or ALK4) and thus inhibit BMP6-mediated signaling (e.g., Smad signaling). In some embodiments, a BMP6 antibody may inhibit BMP6 from binding to a co-receptor and thus inhibit BMP6-mediated signaling (e.g., Smad signaling). In some embodiments, the disclosure relates to a multispecific antibody (e.g., bi-specific antibody), and uses thereof, that binds to BMP6 and further binds to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a multispecific antibody that binds to BMP6 does not bind or does not substantially bind to BMP9 (e.g., binds to BMP9 with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecific antibody that binds to BMP6 does not bind or does not substantially bind to activin A (e.g., binds to activin A with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises a BMP6 antibody and one or more additional antibodies that bind to, for example, one or more ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a combination of antibodies that comprises a BMP6 antibody does not comprise a BMP9 antibody. In some embodiments, a combination of antibodies that comprises a BMP6 antibody does not comprise an activin A antibody.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least BMP10. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least BMP10. As used herein, a BMP10 antibody (or anti-BMP10 antibody) generally refers to an antibody that can bind to BMP10 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting BMP10. In certain embodiments, the extent of binding of a BMP10 antibody to an unrelated, non-BMP10 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to BMP10 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein interaction or binding affinity assay. In certain embodiments, a BMP10 antibody binds to an epitope of BMP10 that is conserved among BMP10 from different species. In certain preferred embodiments, an anti-BMP10 antibody binds to human BMP10. In some embodiments, a BMP10 antibody may inhibit BMP10 from binding to a type I and/or type II receptor (e.g., ActRIIA, ActRIIB, and/or ALK4) and thus inhibit BMP10-mediated signaling (e.g., Smad signaling). In some embodiments, a BMP10 antibody may inhibit BMP10 from binding to a co-receptor and thus inhibit BMP10-mediated signaling (e.g., Smad signaling). In some embodiments, the disclosure relates to a multispecific antibody (e.g., bi-specific antibody), and uses thereof, that binds to BMP10 and further binds to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a multispecific antibody that binds to BMP10 does not bind or does not substantially bind to BMP9 (e.g., binds to BMP9 with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, a multispecific antibody that binds to BMP10 does not bind or does not substantially bind to activin A (e.g., binds to activin A with a K_D of greater than 1×10⁻⁷ M or has relatively modest binding, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises a BMP10 antibody and one or more additional antibodies that bind to, for example, one or more additional ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4). In some embodiments, a combination of antibodies that comprises a BMP10 antibody does not comprise a BMP9 antibody. In some embodiments, a combination of antibodies that comprises a BMP10 antibody does not comprise an activin A antibody.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least ActRIIB. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least ActRIIB. As used herein, an ActRIIB antibody (anti-ActRIIB antibody) generally refers to an antibody that binds to ActRIIB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIB. In certain embodiments, the extent of binding of an anti-ActRIIB antibody to an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to ActRIIB as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-ActRIIB antibody binds to an epitope of ActRIIB that is conserved among ActRIIB from different species. In certain preferred embodiments, an anti-ActRIIB antibody binds to human ActRIIB. In some embodiments, an anti-ActRIIB antibody may inhibit one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) from binding to ActRIIB. In some embodiments, an anti-ActRIIB antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to ActRIIB and one or more ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (e.g., ActRIIA), and/or type I receptor (e.g., ALK4). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises an anti-ActRIIB antibody and one or more additional antibodies that bind to, for example, one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), type I receptors (e.g., ALK4), and/or additional type II receptors (e.g., ActRIIA). It should be noted that ActRIIB has sequence similarity to ActRIIA and therefore antibodies that bind to ActRIIB, in some instances, may also bind to and/or inhibit ActRIIA.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least ActRIIA. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least ActRIIA. As used herein, an ActRIIA antibody (anti-ActRIIA antibody) generally refers to an antibody that binds to ActRIIA with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIA. In certain embodiments, the extent of binding of an anti-ActRIIA antibody to an unrelated, non-ActRIIA protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 10% of the binding of the antibody to ActRIIA as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-ActRIIA antibody binds to an epitope of ActRIIA that is conserved among ActRIIA from different species. In certain preferred embodiments, an anti-ActRIIA antibody binds to human ActRIIA. In some embodiments, an anti-ActRIIA antibody may inhibit one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) from binding to ActRIIA. In some embodiments, an anti-ActRIIA antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to ActRIIA and one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), type I receptor (e.g., ALK4), and/or an additional type II receptor (e.g., ActRIIB). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises an anti-ActRIIA antibody and one or more additional antibodies that bind to, for example, one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), type I receptors (e.g., ALK4), and/or additional type II receptors (e.g., ActRIIB). It should be noted that ActRIIA has sequence similarity to ActRIIB and therefore antibodies that bind to ActRIIA, in some instances, may also bind to and/or inhibit ActRIIB.

In certain aspects, an ActRII-ALK4 antagonist antibody, or combination of antibodies, is an antibody that inhibits at least ALK4. Therefore, in some embodiments, an ActRII-ALK4 antagonist antibody, or combination of antibodies, binds to at least ALK4. As used herein, an ALK4 antibody (anti-ALK4 antibody) generally refers to an antibody that binds to ALK4 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ALK4. In certain embodiments, the extent of binding of an anti-ALK4 antibody to an unrelated, non-ALK4 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the binding of the antibody to ALK4 as measured, for example, by a radioimmunoassay (RIA), Biacore, or other protein-protein interaction or binding affinity assay. In certain embodiments, an anti-ALK4 antibody binds to an epitope of ALK4 that is conserved among ALK4 from different species. In certain preferred embodiments, an anti-ALK4 antibody binds to human ALK4. In some embodiments, an anti-ALK4 antibody may inhibit one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10) from binding to ALK4. In some embodiments, an anti-ALK4 antibody is a multispecific antibody (e.g., bi-specific antibody) that binds to ALK4 and one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), and/or type II receptor (e.g., ActRIIA and/or ActRIIB). In some embodiments, the disclosure relates to combinations of antibodies, and uses thereof, wherein the combination of antibodies comprises an anti-ALK4 antibody and one or more additional antibodies that bind to, for example, one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), and/or type II receptors (e.g., ActRIIA and/or ActRIIB).

The term antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments [see, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894; 5,587,458; and 5,869,046]. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific [see, e.g., EP 404,097; WO 1993/01161; Hudson et al. (2003) Nat. Med. 9:129-134 (2003); and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448]. Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy-chain variable domain or all or a portion of the light-chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody [see, e.g., U.S. Pat. No. 6,248,516]. Antibodies disclosed herein may be polyclonal antibodies or monoclonal antibodies. In certain embodiments, the antibodies of the present disclosure comprise a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme, or enzyme co-factor). In certain preferred embodiments, the antibodies of the present disclosure are isolated antibodies. In certain preferred embodiments, the antibodies of the present disclosure are recombinant antibodies.

The antibodies herein may be of any class. The class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu.

In general, an antibody for use in the methods disclosed herein specifically binds to its target antigen, preferably with high binding affinity. Affinity may be expressed as a K_D value and reflects the intrinsic binding affinity (e.g., with minimized avidity effects). Typically, binding affinity is measured in vitro, whether in a cell-free or cell-associated setting. Any of a number of assays known in the art, including those disclosed herein, can be used to obtain binding affinity measurements including, for example, Biacore, radiolabeled antigen-binding assay (RIA), and ELISA. In some embodiments, antibodies of the present disclosure bind to their target antigens (e.g., ActRIIA, ActRIIB, activin A, activin B, GDF8, GDF11, BMP6, BMP10), with at least a K_D of 1×10⁻⁷ or stronger, 1×10⁻⁸ or stronger, 1×10⁻⁹ or stronger, 1×10⁻¹⁰ or stronger, 1×10⁻¹¹ or stronger, 1×10⁻¹² or stronger, 1×10⁻¹³ or stronger, or 1×10⁻¹⁴ or stronger.

In certain embodiments, K_D is measured by RIA performed with the Fab version of an antibody of interest and its target antigen as described by the following assay. Solution binding affinity of Fabs for the antigen is measured by equilibrating Fab with a minimal concentration of radiolabeled antigen (e.g., ¹²⁵I-labeled) in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol. Biol. 293:865-881]. To establish conditions for the assay, multi-well plates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g., overnight) with a capturing anti-Fab antibody (e.g., from Cappel Labs) and subsequently blocked with bovine serum albumin, preferably at room temperature (approximately 23° C.). In a non-adsorbent plate, radiolabeled antigen are mixed with serial dilutions of a Fab of interest [e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab of interest is then incubated, preferably overnight but the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation, preferably at room temperature for about one hour. The solution is then removed and the plate is washed times several times, preferably with polysorbate 20 and PBS mixture. When the plates have dried, scintillant (e.g., MICROSCINT® from Packard) is added, and the plates are counted on a gamma counter (e.g., TOPCOUNT® from Packard).

According to another embodiment, K_D is measured using surface plasmon resonance assays using, for example a BIACORE®2000 or a BIACORE® 3000 (BIAcore, Inc., Piscataway, N.J.) with immobilized antigen CM5 chips at about 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. For example, an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (about 0.2 μM) before injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at a flow rate of approximately 25 μl/min. Association rates (k_on) and dissociation rates (k_off) are calculated using, for example, a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K_D) is calculated as the ratio k_off/k_on [see, e.g., Chen et al., (1999) J. Mol. Biol. 293:865-881]. If the on-rate exceeds, for example, 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (e.g., excitation=295 nm; emission=340 nm, 16 nm band-pass) of a 20 nM anti-antigen antibody (Fab form) in PBS in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO® spectrophotometer (ThermoSpectronic) with a stirred cuvette.

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein. The nucleic acid and amino acid sequences of human ActRIIA, ActRIIB, ALK4, activin (activin A, activin B, activin C, and activin E), GDF11, GDF8, BMP10, and BMP6, are known in the art. In addition, numerous methods for generating antibodies are well known in the art, some of which are described herein. Therefore, antibody antagonists for use in accordance with this disclosure may be routinely made by the skilled person in the art based on the knowledge in the art and teachings provided herein.

In certain embodiments, an antibody provided herein is a chimeric antibody. A chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855. In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. In general, chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody provided herein is a humanized antibody. A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337; 7,527,791; 6,982,321; and U.S. Pat. No. 7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR (a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498 (describing “resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60 (describing “FR shuffling”); Osbourn et al. (2005) Methods 36:61-68; and Klimka et al. Br. J. Cancer (2000) 83:252-260 (describing the “guided selection” approach to FR shuffling). Human framework regions that may be used for humanization include but are not limited to framework regions selected using the “best-fit” method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296]; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions [see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol., 151:2623]; human mature (somatically mutated) framework regions or human germline framework regions [see, e.g., Almagro and Fransson (2008) Front. Biosci. 13:1619-1633]; and framework regions derived from screening FR libraries [see, e.g., Baca et al., (1997) J. Biol. Chem. 272:10678-10684; and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618].

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel (2008) Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459. For example, human antibodies may be prepared by administering an immunogen (e.g., ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4)) to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals see, for example, Lonberg (2005) Nat. Biotech. 23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describing HuMab® technology); U.S. Pat. No. 7,041,870 (describing K-M MOUSE® technology); and U.S. Patent Application Publication No. 2007/0061900 (describing VelociMouse® technology). Human variable regions from intact antibodies generated by such animals may be further modified, for example, by combining with a different human constant region.

Human antibodies provided herein can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described [see, e.g., Kozbor J. Immunol., (1984) 133: 3001; Brodeur et al. (1987) Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York; and Boerner et al. (1991) J. Immunol., 147: 86]. Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., (2006) Proc. Natl. Acad. Sci. USA, 103:3557-3562. Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue (2006) 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein (2005) Histol. Histopathol., 20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp. Clin. Pharmacol., 27(3):185-91. Human antibodies provided herein may also be generated by isolating Fv clone variable-domain sequences selected from human-derived phage display libraries. Such variable-domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are known in the art and described herein.

For example, antibodies of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. A variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, N.J. and further described, for example, in the McCafferty et al. (1991) Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Marks et al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) in Methods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa, N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al. (2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad. Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods 284(1-2): 119-132.

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. (1994) Ann. Rev. Immunol., 12: 433-455. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen (e.g., ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), and/or type I receptor (e.g., ALK4)) without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. (1993) EMBO J, 12: 725-734. Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

In certain embodiments, an antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies (typically monoclonal antibodies) that have binding specificities for at least two different epitopes (e.g., two, three, four, five, or six or more) on one or more (e.g., two, three, four, five, six or more) antigens.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain/light-chain pairs having different specificities [see, e.g., Milstein and Cuello (1983) Nature 305: 537; International patent publication no. WO 93/08829; and Traunecker et al. (1991) EMBO J. 10: 3655, and U.S. Pat. No. 5,731,168 (“knob-in-hole” engineering)]. Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004A1); cross-linking two or more antibodies or fragments [see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al. (1985) Science, 229: 81]; using leucine zippers to produce bispecific antibodies [see, e.g., Kostelny et al. (1992) J. Immunol., 148(5):1547-1553]; using “diabody” technology for making bispecific antibody fragments [see, e.g., Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448]; using single-chain Fv (sFv) dimers [see, e.g., Gruber et al. (1994) J. Immunol., 152:5368]; and preparing trispecific antibodies (see, e.g., Tutt et al. (1991) J. Immunol. 147: 60. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments. Engineered antibodies with three or more functional antigen-binding sites, including “Octopus antibodies,” are also included herein [see, e.g., US 2006/0025576A1].

In certain embodiments, an antibody disclosed herein is a monoclonal antibody. Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

For example, by using immunogens derived from activin, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols [see, e.g., Antibodies: A Laboratory Manual ed. by Harlow and Lane (1988) Cold Spring Harbor Press: 1988]. A mammal, such as a mouse, hamster, or rabbit, can be immunized with an immunogenic form of the activin polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an activin polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody production and/or level of binding affinity.

Following immunization of an animal with an antigenic preparation of activin, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique [see, e.g., Kohler and Milstein (1975) Nature, 256: 495-497], the human B cell hybridoma technique [see, e.g., Kozbar et al. (1983) Immunology Today, 4:72], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an activin polypeptide, and monoclonal antibodies isolated from a culture comprising such hybridoma cells.

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution, deletion, and/or addition) at one or more amino acid positions.

For example, the present disclosure contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions [e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)] are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in, for example, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom, I. et al. (1986) Proc. Natl. Acad. Sci. USA 83:7059-7063]; Hellstrom, I et al. (1985) Proc. Natl. Acad. Sci. USA 82:1499-1502; U.S. Pat. No. 5,821,337; Bruggemann, M. et al. (1987) J. Exp. Med. 166:1351-1361. Alternatively, non-radioactive assays methods may be employed (e.g., ACTI™, non-radioactive cytotoxicity assay for flow cytometry; CellTechnology, Inc. Mountain View, Calif; and CytoTox 96® non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. (1998) Proc. Natl. Acad. Sci. USA 95:652-656. Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity [see, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402]. To assess complement activation, a CDC assay may be performed [see, e.g., Gazzano-Santoro et al. (1996) J. Immunol. Methods 202:163; Cragg, M. S. et al. (2003) Blood 101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood 103:2738-2743]. FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art [see, e.g., Petkova, S. B. et al. (2006) Intl. Immunol. 18(12):1759-1769]. Antibodies of the present disclosure with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, for example, in U.S. Pat. No. 7,521,541.

In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing interactions between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohistochemistry.

In certain embodiments, amino acid sequence variants of the antibodies and/or the binding polypeptides provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody and/or binding polypeptide. Amino acid sequence variants of an antibody and/or binding polypeptides may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody and/or binding polypeptide, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody and/or binding polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., target-binding (e.g., and activin such as activin E and/or activin C binding).

Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process [see, e.g., Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)], and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described in the art [see, e.g., Hoogenboom et al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, N.J., (2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of the antibody and/or the binding polypeptide that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody-antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is determined to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody and/or binding polypeptide provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody and/or binding polypeptide include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody and/or binding polypeptide may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody and/or binding polypeptide to be improved, whether the antibody derivative and/or binding polypeptide derivative will be used in a therapy under defined conditions.

4. Small Molecule Antagonists

In certain aspects, an ActRII-ALK4 antagonist to be 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 (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure) is a small molecule (ActRII-ALK4 small molecule antagonist), or combination of small molecule antagonists. An ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, may inhibit, for example, one or more ActRII ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), ActRII receptor (ActRIIA and/or ActRIIB), type I receptor (e.g., ALK4), a type II receptor (e.g., ActRIIB and/or ActRIIA), and/or one or more signaling factors. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits signaling mediated by one or more ActRII-ALK4 ligands, for example, as determined in a cell-based assay such as those described herein. As described herein, ActRII-ALK4 small molecule antagonists may be used, alone or in combination with one or more supportive therapies or active agents, to treat, prevent, or reduce the progression rate and/or severity of heart failure), particularly treating, preventing or reducing the progression rate and/or severity of one or more heart failure-associated complications.

In some embodiments, a ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least GDF11, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, a ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least GDF8, optionally further inhibiting one or more of GDF11, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, a ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least activin (activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), optionally further inhibiting one or more of GDF8, GDF11, BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least activin B, optionally further inhibiting one or more of GDF8, GDF11, BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, a ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least BMP6, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), GDF11, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad proteins (e.g., Smads 2 and 3). In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least BMP10, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least ActRIIA, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, BMP10, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least ActRIIB, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, BMP10, ActRIIA, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, inhibits at least ALK4, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, BMP10, ActRIIA, ActRIIB, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, as disclosed herein does not inhibit or does not substantially inhibit BMP9. In some embodiments, an ActRII-ALK4 small molecule antagonist, or combination of small molecule antagonists, as disclosed herein does not inhibit or does not substantially inhibit activin A.

ActRII-ALK4 small molecule antagonists can be direct or indirect inhibitors. For example, an indirect small molecule antagonist, or combination of small molecule antagonists, may inhibit the expression (e.g., transcription, translation, cellular secretion, or combinations thereof) of at least one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), type I receptor (e.g., ALK4), type II receptors (e.g., ActRIIA and/or ActRIIB), and/or one or more downstream signaling components (e.g., Smads). Alternatively, a direct small molecule antagonist, or combination of small molecule antagonists, may directly bind to and inhibit, for example, one or more one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), type I receptor (e.g., ALK4), type II receptors (e.g., ActRIIA and/or ActRIIB), and/or one or more downstream signaling components (e.g., Smads). Combinations of one or more indirect and one or more direct ActRII-ALK4 small molecule antagonists may be used in accordance with the methods disclosed herein.

Binding small-molecule antagonists of the present disclosure may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general, small molecule antagonists of the disclosure are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molecules that are capable of binding, preferably specifically, to a polypeptide as described herein. These small molecule antagonists may be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for screening organic small-molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., international patent publication Nos. WO00/00823 and WO00/39585).

Binding organic small molecules of the present disclosure may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, and acid chlorides.

5. Polynucleotide Antagonists

In certain aspects, an ActRII-ALK4 antagonist to be 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 (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) or one or more complications of heart failure) is a polynucleotide (ActRII-ALK4 polynucleotide antagonist), or combination of polynucleotides. An ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, may inhibit, for example, one or more ActRII-ALK4 ligands (e.g., activin A, activin B, GDF8, GDF11, BMP6, BMP10), type I receptors (e.g., ALK4), type II receptors (e.g., ActRIIA and/or ActRIIB), and/or downstream signaling component (e.g., Smads). In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits signaling mediated by one or more ActRII-ALK4 ligands, for example, as determined in a cell-based assay such as those described herein. As described herein, ActRII-ALK4 polynucleotide antagonists may be used, alone or in combination with one or more supportive therapies or active agents, to treat, prevent, or reduce the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies)), particularly treating, preventing or reducing the progression rate and/or severity of one or more heart failure-associated complications

In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least GDF11, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, BMP10, ActRIIA ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least GDF8, optionally further inhibiting one or more of GDF11, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least activin (activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), optionally further inhibiting one or more of GDF8, GDF11, BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least activin B, optionally further inhibiting one or more of GDF8, GDF11, BMP6, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least BMP6, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), GDF11, BMP10, ActRIIA, ActRIIB, ALK4, and one or more Smad proteins signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least BMP10, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, ActRIIA, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least ActRIIA, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, BMP10, ActRIIB, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least ActRIIB, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, ActRIIA, BMP10, ALK4, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, inhibits at least ALK4, optionally further inhibiting one or more of GDF8, activin (e.g., activin A, activin B, activin C, activin E, activin AB, activin AC, activin BC, activin AE and/or activin BE), BMP6, GDF11, ActRIIA, ActRIIB, BMP10, and one or more Smad signaling factors. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, as disclosed herein does not inhibit or does not substantially inhibit BMP9. In some embodiments, an ActRII-ALK4 polynucleotide antagonist, or combination of polynucleotide antagonists, as disclosed herein does not inhibit or does not substantially inhibit activin A.

In some embodiments, the polynucleotide antagonists of the disclosure may be an antisense nucleic acid, an RNAi molecule [e.g., small interfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)], an aptamer and/or a ribozyme. The nucleic acid and amino acid sequences of human GDF11, GDF8, activin (activin A, activin B, activin C, and activin E), BMP6, ActRIIA, ActRIIB, BMP10, ALK4, and Smad signaling factors are known in the art. In addition, many different methods of generating polynucleotide antagonists are well known in the art. Therefore, polynucleotide antagonists for use in accordance with this disclosure may be routinely made by the skilled person in the art based on the knowledge in the art and teachings provided herein.

Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed, for example, in Okano (1991) J. Neurochem. 56:560; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple-helix formation is discussed in, for instance, Cooney et al. (1988) Science 241:456; and Dervan et al., (1991) Science 251:1300. The methods are based on binding of a polynucleotide to a complementary DNA or RNA. In some embodiments, the antisense nucleic acids comprise a single-stranded RNA or DNA sequence that is complementary to at least a portion of an RNA transcript of a gene disclosed herein. However, absolute complementarity, although preferred, is not required.

A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids of a gene disclosed herein, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Polynucleotides that are complementary to the 5′ end of the message, for example, the 5′-untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′-untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature 372:333-335]. Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a gene of the disclosure, could be used in an antisense approach to inhibit translation of an endogenous mRNA. Polynucleotides complementary to the 5′-untranslated region of the mRNA should include the complement of the AUG start codon. Antisense polynucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the methods of the present disclosure. Whether designed to hybridize to the 5′-, 3′- or coding region of an mRNA of the disclosure, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

In one embodiment, the antisense nucleic acid of the present disclosure is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of a gene of the disclosure. Such a vector would contain a sequence encoding the desired antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding desired genes of the instant disclosure, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region [see, e.g., Benoist and Chambon (1981) Nature 290:304-310], the promoter contained in the 3′ long-terminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al. (1980) Cell 22:787-797], the herpes thymidine promoter [see, e.g., Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], and the regulatory sequences of the metallothionein gene [see, e.g., Brinster, et al. (1982) Nature 296:39-42].

In some embodiments, the polynucleotide antagonists are interfering RNA (RNAi) molecules that target the expression of one or more of: GDF11, GDF8, activin (activin A, activin B, activin C, and activin E), BMP6, ActRIIA, ActRIIB, BMP10, ALK4, and Smad signaling factors. RNAi refers to the expression of an RNA which interferes with the expression of the targeted mRNA. Specifically, RNAi silences a targeted gene via interacting with the specific mRNA through a siRNA (small interfering RNA). The ds RNA complex is then targeted for degradation by the cell. An siRNA molecule is a double-stranded RNA duplex of 10 to 50 nucleotides in length, which interferes with the expression of a target gene which is sufficiently complementary (e.g. at least 80% identity to the gene). In some embodiments, the siRNA molecule comprises a nucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the nucleotide sequence of the target gene.

Additional RNAi molecules include short-hairpin RNA (shRNA); also, short-interfering hairpin and microRNA (miRNA). The shRNA molecule contains sense and antisense sequences from a target gene connected by a loop. The shRNA is transported from the nucleus into the cytoplasm, and it is degraded along with the mRNA. Pol III or U6 promoters can be used to express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002) 16:948-958, 2002] have used small RNA molecules folded into hairpins as a means to affect RNAi. Accordingly, such short-hairpin RNA (shRNA) molecules are also advantageously used in the methods described herein. The length of the stem and loop of functional shRNAs varies; stem lengths can range anywhere from about 25 to about 30 nt, and loop size can range between 4 to about 25 nt without affecting silencing activity. While not wishing to be bound by any particular theory, it is believed that these shRNAs resemble the double-stranded RNA (dsRNA) products of the DICER RNase and, in any event, have the same capacity for inhibiting expression of a specific gene. The shRNA can be expressed from a lentiviral vector. An miRNA is a single-stranded RNA of about 10 to 70 nucleotides in length that are initially transcribed as pre-miRNA characterized by a “stem-loop” structure, which are subsequently processed into mature miRNA after further processing through the RISC.

Molecules that mediate RNAi, including without limitation siRNA, can be produced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199, 2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase (Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-stranded RNA using a nuclease such as E. coli RNase III (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002).

According to another aspect, the disclosure provides polynucleotide antagonists including but not limited to, a decoy DNA, a double-stranded DNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA, a double-stranded RNA, a molecule capable of generating RNA interference, or combinations thereof.

In some embodiments, the polynucleotide antagonists of the disclosure are aptamers. Aptamers are nucleic acid molecules, including double-stranded DNA and single-stranded RNA molecules, which bind to and from tertiary structures that specifically bind to a target molecule. The generation and therapeutic use of aptamers are well established in the art (see, e.g., U.S. Pat. No. 5,475,096). Additional information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. Nucleic acid aptamers are selected using methods known in the art, for example via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules as described in, e.g., U.S. Pat. Nos. 5,475,096; 5,580,737; 5,567,588; 5,707,796; 5,763,177; 6,011,577; and 6,699,843. Another screening method to identify aptamers is described in U.S. Pat. No. 5,270,163. The SELEX process is based on the capacity of nucleic acids for forming a variety of two- and three-dimensional structures, as well as the chemical versatility available within the nucleotide monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric, including other nucleic acid molecules and polypeptides. Molecules of any size or composition can serve as targets. The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve desired binding affinity and selectivity. Starting from a mixture of nucleic acids, which can comprise a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding; partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; dissociating the nucleic acid-target complexes; amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids. The steps of binding, partitioning, dissociating and amplifying are repeated through as many cycles as desired to yield nucleic acid ligands which bind with high affinity and specificity to the target molecule.

Typically, such binding molecules are separately administered to the animal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but such binding molecules can also be expressed in vivo from polynucleotides taken up by a host cell and expressed in vivo [see, e.g., Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)].

6. Heart Failure

In part, the present disclosure relates to a method of treating heart failure comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure relates to a method of treating dilated cardiomyopathy. In some embodiments, the disclosure relates to a method of treating heart failure associated with a muscular dystrophy (e.g., DMD). In some embodiments, the disclosure relates to a method of treating heart failure associated with a muscle wasting disease. In some embodiments, the disclosure relates to a method of treating a genetic cardiomyopathy. In some embodiments, the disclosure relates to a method of treating heart failure associated with Duchenne muscular dystrophy (DMD). In some embodiments, the disclosure relates to a method of treating heart failure associated with Limb girdle muscular dystrophy (LGMD). In some embodiments, the disclosure relates to a method of treating heart failure associated with Friedreich's ataxia. In some embodiments, the disclosure relates to a method of treating heart failure associated with Myotonic dystrophy. In some embodiments, the method relates to heart failure patients that have Dilated cardiomyopathy (DCM). In some embodiments, the disclosure relates to a method of treating Hypertrophic cardiomyopathy (HCM). In some embodiments, the disclosure relates to a method of treating Arrhythmogenic cardiomyopathy (AC). In some embodiments, the disclosure relates to a method of treating Left ventricular noncompaction cardiomyopathy (LVNC). In some embodiments, the disclosure relates to a method of treating Restrictive cardiomyopathy (RC).

These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans. The terms “subject,” an “individual,” or a “patient” are interchangeable throughout the specification and refer to either a human or a non-human animal. These terms include mammals, such as humans, non-human primates, laboratory animals, livestock animals (including bovines, porcines, camels, etc.), companion animals (e.g., canines, felines, other domesticated animals, etc.) and rodents (e.g., mice and rats). In particular embodiments, the patient, subject or individual is a human.

The terms “treatment”, “treating”, “alleviating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect, and may also be used to refer to improving, alleviating, and/or decreasing the severity of one or more clinical complication of a condition being treated (e.g., heart failure). The effect may be prophylactic in terms of completely or partially delaying the onset or recurrence of a disease, condition, or complications thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition. “Treatment” as used herein covers any treatment of a disease or condition of a mammal, particularly a human. As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the onset of the disease or condition, relative to an untreated control sample.

In general, treatment or prevention of a disease or condition as described in the present disclosure (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) is achieved by administering one or more ActRII-ALK4 antagonists of the disclosure in an “effective amount”. An effective amount of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A “therapeutically effective amount” of an agent of the present disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

The main terminology used to describe HF is based on measurement of left ventricular ejection fraction (LVEF). HF comprises a wide range of patients (Table 1). Some patients have normal LVEF, which is typically considered as ≥50% and is referred to as HF with preserved ejection fraction (HFpEF). Other patients have HF with reduced LVEF (HFrEF), which is typically considered to be <40%. Patients with an LVEF in the range of between about 40% and about 49% represent a “grey area”, which is sometimes defined as HF with mid-range ejection fraction (HFmrEF). Sometimes these patients in the “grey area” are identified as having HFrEF, depending on the clinician. Differentiation of patients with HF based on LVEF is important due to different underlying etiologies, demographics, co-morbidities and response to therapies. Most clinical trials published after 1990 selected patients based on LVEF (usually measured using echocardiography, a radionuclide technique or cardiac magnetic resonance (CMR)), and to the best of our knowledge, it is only in patients with HFrEF that therapies have been shown to reduce both morbidity and mortality.

TABLE 1
Definition of heart failure by left ventricular ejection fraction analysis
Type of
HF	HFrEF	HFmrEF	HFpEF
Criteria	1	Symptoms ± Signs	Symptoms ± Signs	Symptoms ± Signs
	2	LVEF <40%	LVEF 40-49%	LVEF ≥50%
	3	—	1. Elevated levels of	1. Elevated levels of
			natriuretic peptides	natriuretic peptides
			2. At least one additional	2. At least one additional
			criterion:	criterion:
			a. relevant structural heart	a. relevant structural heart
			disease (LVH and/or LAE)	disease (LVH and/or LAE)
			b. diastolic dysfunction	b. diastolic dysfunction
Symptoms: e.g., breathlessness, ankle swelling and fatigue
Signs: e.g., elevated jugular venous pressure, pulmonary crackles and peripheral edema. Signs may not be present in the early stages of HF (especially in HFpEF) and in patients treated with diuretics.
Symptoms and signs are caused by a structural and/or functional cardiac abnormality.
HF = heart failure;
HFmrEF = heart failure with mid-range ejection fraction;
HFpEF = heart failure with preserved ejection fraction;
HFrEF = heart failure with reduced ejection fraction;
LAE = left atrial enlargement;
LVEF = left ventricular ejection fraction;
LVH = left ventricular hypertrophy;

In certain aspects, the disclosure relates to a method of treating, preventing, or reducing the progression rate and/or severity of heart failure with preserved ejection fraction (HFpEF) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure relates to a method of treating a patient that has normal LVEF. In some embodiments, the disclosure relates to a method of treating a patient having normal LVEF and an LVEF of ≥50%. In some embodiments, the disclosure relates to a method of treating a patient having normal LVEF and HF associated with preserved ejection fraction (HFpEF). In some embodiments, the disclosure relates to a method of treating a patient having HFpEF and elevated levels of natriuretic peptides. In some embodiments, the disclosure relate to treating a patient having HFpEF, elevated levels of natriuretic peptides, and a structural heart disease and/or diastolic dysfunction.

In certain aspects, the disclosure relates to a method of treating, preventing, or reducing the progression rate and/or severity of heart failure with reduced ejection fraction (HFrEF) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure relates to a method of treating a patient having reduced LVEF. In some embodiments, the disclosure relates to a method of treating a patient with reduced LVEF and an LVEF of <40%. In some embodiments, the disclosure relates to a method of treating a patient with reduced LVEF and HF associated with reduced ejection fraction (HFrEF).

In certain aspects, the disclosure relates to a method of treating, preventing, or reducing the progression rate and/or severity of heart failure with mid-range ejection fraction (HFmrEF) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure relates to a method of treating a patient has mid-range LVEF. In some embodiments, the disclosure relates to a method of treating a patient with mid-range LVEF and an LVEF of between about 40% and about 49%. In some embodiments, the disclosure relates to treating a patient with mid-range LVEF and HF associated with mid-range ejection fraction (HFmrEF). In some embodiments, the disclosure relates to a method of treating a patient having HmrEF and elevated levels of natriuretic peptides. In some embodiments, the disclosure relates to a method of treating a patient having HFmrEF and elevated levels of natriuretic peptides, and a structural heart disease and/or diastolic dysfunction.

Diagnosis of HFpEF can be more challenging than a diagnosis of HFrEF. Patients with HFpEF generally do not have a dilated LV, but instead often have an increase in LV wall thickness and/or increased left atrial (LA) size as a sign of increased filling pressures. Most have additional ‘evidence’ of impaired LV filling or suction capacity, also classified as diastolic dysfunction, which is generally accepted as the likely cause of HF in these patients (hence the term ‘diastolic HF’). However, most patients with HFrEF (previously referred to as ‘systolic HF’) also have diastolic dysfunction, and subtle abnormalities of systolic function have been shown in patients with HFpEF. Hence the preference for stating preserved or reduced LVEF over preserved or reduced ‘systolic function’.

In previous guidelines it was acknowledged that a grey area exists between HFrEF and HFpEF. These patients have an LVEF that ranges from 40 to 49%, hence the term HFmrEF. Patients with HFmrEF most likely have primarily mild systolic dysfunction, but with features of diastolic dysfunction.

Patients without detectable LV myocardial disease may have other cardiovascular causes for HF (e.g., pulmonary hypertension, valvular heart disease, etc.). Patients with non-cardiovascular pathologies (e.g., anemia, pulmonary, renal or hepatic disease) may have symptoms similar or identical to those of HF and each may complicate or exacerbate the HF syndrome.

The NYHA functional classification (Table 2) has been used to describe the severity of symptoms and exercise intolerance. However, symptom severity correlates poorly with many measures of LV function; although there is a clear relationship between the severity of symptoms and survival, patients with mild symptoms may still have an increased risk of hospitalization and death. Sometimes the term ‘advanced HF’ is used to characterize patients with severe symptoms, recurrent decompensation and severe cardiac dysfunction.

TABLE 2
New York Heart Association (NYHA) functional classification of HF
based on severity of symptoms and physical activity
Class I   No limitation of physical activity. Ordinary physical activity
          does not cause undue breathlessness, fatigue, or palpitations.
Class II  Slight limitation of physical activity. Comfortable at rest,
          but ordinary physical activity results in undue breathlessness,
          fatigue, or palpitations.
Class III Marked limitation of physical activity. Comfortable at rest,
          but less than ordinary physical activity results in undue
          breathlessness, fatigue, or palpitations.
Class IV  Unable to carry on any physical activity without discomfort.
          Symptoms at rest can be present If any physical activity is
          undertaken, discomfort is increased.

In some embodiments, the disclosure relates to a method of treating a patient having NYHA Class I HF. In some embodiments, a patient with NYHA Class I HF has no limitation of physical activity. In some embodiments, a patient with NYHA Class I HF experiences physical activity that does not cause undue breathlessness, fatigue, and/or palpitations. In some embodiments, the disclosure relates to a method of treating a patient having NYHA Class II HF. In some embodiments, a patient with NYHA Class II HF has slight limitation of physical activity. In some embodiments, a patient with NYHA Class II HF experiences ordinary physical activity resulting in undue breathlessness, fatigue, or palpitations. In some embodiments, the disclosure relates to a method of treating a patient having NYHA Class III HF. In some embodiments, a patient with NYHA Class III HF has marked limitation of physical activity. In some embodiments, a patient with NYHA Class III HF experiences less than ordinary physical activity resulting in undue breathlessness, fatigue, or palpitations. In some embodiments, the disclosure relates to a method of treating a patient having NYHA Class IV HF. In some embodiments, a patient with NYHA Class IV HF is unable to carry on any physical activity without discomfort. In some embodiments, a patient with NYHA Class IV HF experiences symptoms at rest, as well as when any physical activity is undertaken, discomfort is increased.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method improves the patient's NYHA functional heart failure Class. In some embodiments, the method relates to reducing the patient's NYHA Class from Class IV to Class III. In some embodiments, the method relates to reducing the patient's NYHA Class from Class IV to Class II. In some embodiments, the method relates to reducing the patient's NYHA Class from Class IV to Class I. In some embodiments, the method relates to reducing the patient's NYHA Class from Class III to Class II. In some embodiments, the method relates to reducing the patient's NYHA Class from Class III to Class I. In some embodiments, the method relates to reducing the patient's NYHA Class from Class II to Class I.

The American College of Cardiology Foundation/American Heart Association (ACCF/AHA) classification describes stages of HF development based on structural changes and symptoms (Table 3). The ACC/AHA classification system places emphasis on staging and development of disease, similar to the approach commonly used in oncology. These HF stages progress from antecedent risk factors (stage A) to the development of subclinical cardiac dysfunction (stage B), then symptomatic HF (stage C), and finally, end-stage refractory disease (stage D). ACC/AHA stages are progressive from stage A to stage D.

TABLE 3
American College of Cardiology Foundation/American Heart
Association (ACCF/AHA) stages of heart failure
A At high risk for HF but without structural heart disease or
  symptoms of HF.
B Structural heart disease but without signs or symptoms of HF.
C Structural heart disease with prior or current symptoms of HF.
D Refractory HF requiring specialized interventions.

In some embodiments, the disclosure relates to a method of treating a patient having ACCF/AHA Stage A HF. In some embodiments, a patient with ACCF/AHA Stage A HF is at high risk for HF but without structural heart disease or symptoms of HF. In some embodiments, the disclosure relates to a method of treating a patient having ACCF/AHA Stage B HF. In some embodiments, a patient with Stage B HF has structural heart disease but without known signs or symptoms of HF. In some embodiments, the disclosure relates to a method of treating a patient having ACCF/AHA Stage C HF. In some embodiments, a patient with ACCF/AHA Stage C HF has structural heart disease with prior or current symptoms of HF. In some embodiments, the disclosure relates to a method of treating a patient having ACCF/AHA Stage D HF. In some embodiments, a patient with ACCF/AHA Stage D HF has refractory HF requiring specialized interventions.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method improves the patient's ACCF/AHA stage of heart failure. In some embodiments, the method relates to reducing the patient's ACCF/AHA Stage from Stage D to Stage C. In some embodiments, the method relates to reducing the patient's ACCF/AHA Stage from Stage D to Stage B. In some embodiments, the method relates to reducing the patient's ACCF/AHA Stage from Stage D to Stage A. In some embodiments, the method relates to reducing the patient's ACCF/AHA Stage from Stage C to Stage B. In some embodiments, the method relates to reducing the patient's ACCF/AHA Stage from Stage C to Stage A. In some embodiments, the method relates to reducing the patient's ACCF/AHA Stage from Stage B to Stage A.

The Killip classification may be used to describe the severity of the patient's condition in the acute setting after myocardial infarction. Patients with HF complicating acute myocardial infarction (AMI) can be classified according to Killip and Kimball into the classes shown in Table 4.

TABLE 4
Killip Classification of HF complicating AMI
Class I   No clinical signs of HF
Class II  HF with rales and S3 gallop
Class III With frank acute pulmonary edema
Class IV  Cardiogenic shock, hypotension (SBP, 90 mmHg)
          and evidence of peripheral vasoconstriction such
          as oliguria, cyanosis and diaphoresis

In some embodiments, the disclosure relates to a method of treating a patient having Killip Class I HF complicating AMI. In some embodiments, a patient with Killip Class I HF complicating AMI has no clinical signs of HF. In some embodiments, the disclosure relates to a method of treating a patient having Killip Class II HF complicating AMI. In some embodiments, a patient with Killip Class II HF complicating AMI has HF with rales and S3 gallop. In some embodiments, the disclosure relates to a method of treating a patient having Killip Class III HF complicating AMI. In some embodiments, a patient with Killip Class III HF complicating AMI has frank acute pulmonary edema. In some embodiments, the disclosure relates to a methods of treating a patient having Killip Class IV HF complicating AMI. In some embodiments, a patient with Killip Class IV HF complicating AMI has cardiogenic shock, hypotension (e.g., SBP, 90 mmHg) and evidence of peripheral vasoconstriction such as oliguria, cyanosis and diaphoresis.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method improves the patient's Killip HF Classification. In some embodiments, the method relates to reducing the patient's Killip Class from Class IV to Class III. In some embodiments, the method relates to reducing the patient's Killip Class from Class IV to Class II. In some embodiments, the method relates to reducing the patient's Killip Class from Class IV to Class I. In some embodiments, the method relates to reducing the patient's Killip Class from Class III to Class II. In some embodiments, the method relates to reducing the patient's Killip Class from Class III to Class I. In some embodiments, the method relates to reducing the patient's Killip Class from Class II to Class I.

The Framingham criteria for diagnosis of heart failure (Table 5) requires presence of at least two major criteria, or at least one major and two minor criteria. Although these criteria have served as a gold reference standard for decades, they are largely predicated on the presence of congestion at rest. Importantly, this clinical feature is often absent in ambulatory patients who have well-compensated HF, or in patients with HF who develop abnormal hemodynamics exclusively during exercise. Therefore, despite being highly specific, the Framingham criteria tend to have a poor sensitivity for the diagnosis of HF.

TABLE 5
Framingham criteria for diagnosis of heart failure
Major    Paroxysmal nocturnal dyspnea or orthopnea
criteria Jugular vein distension
         Rales
         Radiographic cardiomegaly
         Acute pulmonary edema
         S3 gallop
         Increased venous pressure greater than 16 cm of water
         Circulation time greater than or equal to 25 seconds
         Hepatojugular reflex
         Weight loss greater than or equal to 4.5 kg in 5 days in
         response to treatment
Minor    Bilateral ankle edema
criteria Nocturnal cough
         Dyspnea on ordinary exertion
         Hepatomegaly
         Pleural effusion
         Decrease in vital capacity by ⅓ from maximum recorded
         Tachycardia (heart rate greater than 120/min)

In some embodiments, the disclosure relates to a methods of treating a patient having one or more major Framingham criteria for diagnosis of HF. In some embodiments, a patient has one or more of paroxysmal nocturnal dyspnea or orthopnea, jugular vein distension, rales, radiographic cardiomegaly, acute pulmonary edema, S3 gallop, increased venous pressure greater than 16 cm of water, circulation time greater than or equal to 25 seconds, hepatojugular reflex, and weight loss greater than or equal to 4.5 kg in 5 days in response to treatment. In some embodiments, the disclosure relates to a methods of treating a patient having one or more minor Framingham criteria for diagnosis of HF. In some embodiments, a patient has one or more of bilateral ankle edema, nocturnal cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion, decrease in vital capacity by ⅓ from maximum recorded, and tachycardia (heart rate greater than 120/min). In some embodiments, a patient has at least two Framingham major criteria. In some embodiments, a patient has at least one major Framingham criteria and at least two minor Framingham criteria.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method reduces the number of Framingham criteria for heart failure that the patient has. In some embodiments, the method relates to decreasing the number of major Framingham criteria for heart failure that the patient has. In some embodiments, the method relates to decreasing the number of minor Framingham criteria for heart failure that the patient has.

There are many known symptoms and signs of heart failure that a medical professional may look for regarding a diagnosis of heart failure. Some symptoms may be non-specific and do not, therefore, help discriminate between HF and other problems. Symptoms and signs of HF due to fluid retention may resolve quickly with diuretic therapy. Signs, such as elevated jugular venous pressure and displacement of the apical impulse, may be more specific, but are harder to detect and have poor reproducibility. HF is unusual in an individual with no relevant medical history (e.g., a potential cause of cardiac damage), whereas certain features, particularly previous myocardial infarction, greatly increase the likelihood of HF in a patient with appropriate symptoms and signs. Symptoms and signs are important in monitoring a patient's response to treatment and stability over time. Persistence of symptoms despite treatment usually indicates the need for additional therapy, and worsening of symptoms is a serious development (placing the patient at risk of urgent hospital admission and death) and merits prompt medical attention.

TABLE 6
Signs and Symptoms of Heart Failure
Symptoms                         Signs
Typical                          More specific
Breathlessness                   Elevated jugular venous pressure
Orthopnea                        Hepatojugular reflux
Paroxysmal nocturnal dyspnea     Third heart sound (gallop rhythm)
Reduced exercise tolerance       Laterally displaced apical impulse
Fatigue, tiredness, increased time to recover
after exercise
Ankle swelling
Less typical                     Less specific
Nocturnal cough                  Weight gain (>2 kg/week)
Wheezing                         Weight loss (in advanced HF)
Bloated feeling                  Tissue wasting (cachexia)
Loss of appetite                 Cardiac murmur
Confusion (especially in the elderly) Peripheral edema (ankle, sacral, scrotal)
Depression                       Pulmonary crepitations
Palpitations                     Reduced air entry and dullness to
Dizziness                        percussion at lung bases (pleural effusion)
Syncope                          Tachycardia
Bendopnea                        Irregular pulse
                                 Tachypnoea
                                 Cheyne Stokes respiration
                                 Hepatomegaly
                                 Ascites
                                 Cold extremities
                                 Oliguria
                                 Narrow pulse pressure

In some embodiments, the disclosure relates to a method of treating a patient having one or more typical and/or less typical symptoms of HF. In some embodiments, the disclosure relates to a method of treating a patient having one or more specific and/or less specific signs of HF. In some embodiments, the disclosure relates to treating a patient having one or more typical symptoms, less typical symptoms, specific signs, and/or less specific signs of HF. In some embodiments, the disclosure relates to a method treating a patient having one or more typical symptoms of HF. In some embodiments, a patient has one or more symptoms selected from the group consisting of breathlessness, orthopnea, paroxysmal nocturnal dyspnea, reduced exercise tolerance, fatigue, tiredness, increased time to recover after exercise, and ankle swelling. In some embodiments, a patient has one or more less typical symptoms of HF. In some embodiments, a patient has one or more less typical symptoms selected from the group consisting of nocturnal cough, wheezing, bloated feeling, loss of appetite, confusion (especially in the elderly), depression, palpitations, dizziness, syncope, and bendopnea. In some embodiments, a patient has one or more signs of HF. In some embodiments, a patient has one or more signs of HF selected from the group consisting of elevated jugular venous pressure, hepatojugular reflux, third heart sound (gallop rhythm), laterally displaced apical impulse. In some embodiments, a patient has one or more less specific signs of HF. In some embodiments, a patient has one or more less specific signs of HF selected from the group consisting of weight gain (>2 kg/week), weight loss (in advanced HF), tissue wasting (cachexia), cardiac murmur, peripheral edema (ankle, sacral, scrotal), pulmonary crepitations, reduced air entry and dullness to percussion at lung bases (pleural effusion), tachycardia, irregular pulse, tachypnoea, Cheyne Stokes respiration, hepatomegaly, ascites, cold extremities, oliguria, and narrow pulse pressure.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method reduces the number of signs and/or symptoms of heart failure that the patient has. In some embodiments, the method relates to decreasing the number of signs of heart failure that the patient has. In some embodiments, the method relates to decreasing the number of symptoms of heart failure that the patient has.

Genetic Cardiomyopathies

Genetic cardiomyopathies are classically divided into dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy (AC), and restrictive cardiomyopathy (RCM), among others, each of which may be the cause of an HF syndrome. There can be extensive overlap between these phenotypes; for example, HCM, left ventricular noncompaction cardiomyopathy (LVNC), and/or AC may progress into a dilated ventricle with systolic dysfunction and hence the appearance of DCM. In genetic cardiomyopathy, as in other forms of HF, advanced imaging offers refinement of a structurally based classification along with functional information to complement the morphological phenotype, providing insight into contractility, diastolic function, strain, synchrony, fibrosis, and energetics. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has a genetic cardiomyopathy. In some embodiments, the method relates to treating dilated cardiomyopathy in a patient having a genetic cardiomyopathy. In some embodiments, the method relates to treating hypertrophic cardiomyopathy in a patient having a genetic cardiomyopathy. In some embodiments, the method relates to treating arrhythmogenic cardiomyopathy in a patient having a genetic cardiomyopathy. In some embodiments, the method relates to treating left ventricular noncompaction cardiomyopathy in a patient having a genetic cardiomyopathy. In some embodiments, the method relates to treating restrictive cardiomyopathy in a patient having a genetic cardiomyopathy.

Genetic cardiomyopathies represent a small proportion of HF overall, although this can vary strongly by age and population. In the pediatric population with HF, a familial and presumed monogenic origin is frequently identified. In younger adults with HF, the prevalence of genetic disease is also high. Similarly, in idiopathic DCM in adults, the proportion of familial disease found upon family screening is high, typically at around >30%. Susceptibility to HF is also heritable as a complex trait, and having a parent with HF who is <75 years of age was found to be a significant the risk factor for development of HF.

Several common characteristics of genetic cardiomyopathies have emerged. First, different variants within an individual gene can produce contrasting phenotypes. For example, mutations in the gene encoding the sarcomeric protein cardiac troponin I (TNNI3) may cause an HCM, DCM, or RCM phenotype. Importantly, in almost all instances, each specific mutation consistently produces the same qualitative phenotype (e.g., a given variant causes either DCM or HCM but not both). However, there is substantial quantitative variability in a given cardiomyopathy phenotype, even when the disease gene and allele are the same, which is referred to as phenotypic heterogeneity. Second, each of the cardiomyopathy phenotypes is caused by one of numerous genetic mutations (genetic heterogeneity). For example, mutations in more than 50 genes can cause a DCM phenotype (locus heterogeneity), and within these genes, numerous different pathogenic mutations are described (allelic heterogeneity). Many mutations therefore are rare and frequently specific to an individual family, with few hot spots or common mutations. The consequence of this heterogeneity is that frequently, testing a patient for only known alleles is not effective as a diagnostic test, and systematic sequencing is typically needed instead. Furthermore, given the high frequency of rare variants in the human genome, the pathogenicity of a missense variant identified in a proband must be validated. Third, genetic cardiomyopathies demonstrate variable penetrance (e.g., the proportion of individuals carrying a pathogenic mutation who display a phenotype) even within the same family. Expressivity (e.g., the severity of a phenotype that develops in a patient with a pathogenic mutation) is also highly variable, meaning the clinical presentation, disease course, and outcome can differ dramatically within an affected family.

Muscle Wasting Diseases

Muscle wasting refers to the progressive loss of muscle mass and/or to the progressive weakening and degeneration of muscles, including the skeletal or voluntary muscles which control movement, cardiac muscles which control the heart (cardiomyopathics), and smooth muscles. Chronic muscle wasting is a chronic condition (i.e. persisting over a long period of time) characterized by progressive loss of muscle mass, weakening and degeneration of muscle.

The loss of muscle mass that occurs during muscle wasting can be characterized by muscle protein degradation by catabolism. Protein catabolism occurs because of an unusually high rate of protein degradation, an unusually low rate of protein synthesis, or a combination of both. Muscle protein catabolism, whether caused by a high degree of protein degradation or a low degree of protein synthesis, leads to a decrease in muscle mass and to muscle wasting.

Muscle wasting is associated with chronic, neurological, genetic or infectious pathologies, diseases, illnesses or conditions. These include muscular dystrophies (e.g., Becker muscular dystrophy (BMD), Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), and Oculopharyngeal muscular dystrophy (OPMD)); muscle atrophies such as Post-Polio Muscle Atrophy (PPMA); cachexias such as cardiac cachexia, AIDS cachexia, and cancer cachexia, malnutrition, leprosy, diabetes, renal disease, Chronic Obstructive Pulmonary Disease (COPD), cancer, end stage renal failure, sarcopenia, emphysema, osteomalacia, HIV infection, and AIDS.

In addition, other circumstances and conditions are linked to and can cause muscle wasting. These include chronic lower back pain, advanced age, central nervous system (CNS) injury, peripheral nerve injury, spinal cord injury, chemical injury, central nervous system (CNS) damage, peripheral nerve damage, spinal cord damage, chemical damage, burns, disuse deconditioning that occurs when a limb is immobilized, long term hospitalization due to illness or injury, and alcoholism.

Muscle wasting, if left unabated, can have dire health consequences. For example, the changes that occur during muscle wasting can lead to a weakened physical state that is detrimental to an individual's health, resulting in increased susceptibility to infraction and poor performance status. In addition, muscle wasting is a strong predictor of morbidity and mortality in patients suffering from cachexia and AIDS. Innovative approaches are urgently needed at both the basic science and clinical levels to prevent and treat muscle wasting, in particular chronic muscle wasting.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has a muscle wasting disease. In some embodiments, the method relates to treating a patient with HFrEF who has one or more muscle wasting diseases. In some embodiments, the disclosure relates to a method of treating a patient that has a muscle wasting disease that is a muscular dystrophy. In some embodiments, the disclosure relates to a method of treating a patient that has one or more muscular dystrophies selected from the group consisting of Becker muscular dystrophy (BMD), Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), and Oculopharyngeal muscular dystrophy (OPMD). In some embodiments, the disclosure relates to a method of treating a patient that has one or more muscle atrophies such as Post-Polio Muscle Atrophy (PPMA). In some embodiments, the disclosure relates to a method of treating a patient that has one or more cachexias selected from the group consisting of cardiac cachexia, AIDS Cachexia and cancer cachexia. In some embodiments, the disclosure relates to a method of treating a patient that has malnutrition. In some embodiments, the disclosure relates to a method of treating a patient that has Leprosy. In some embodiments, the disclosure relates to a method of treating a patient that has diabetes. In some embodiments, the disclosure relates to a method of treating a patient that has renal disease. In some embodiments, the disclosure relates to a method of treating a patient that has Chronic Obstructive Pulmonary Disease (COPD). In some embodiments, the disclosure relates to a method of treating a patient that has cancer. In some embodiments, the disclosure relates to a method of treating a patient that has end stage renal failure. In some embodiments, the disclosure relates to a method of treating a patient that has sarcopenia. In some embodiments, the disclosure relates to a method of treating a patient that has osteomalacia. In some embodiments, the disclosure relates to a method of treating a patient that has HIV Infection. In some embodiments, the disclosure relates to a method of treating a patient that has AIDS. In some embodiments, the disclosure relates to a method of treating a patient that has a cardiomyopathy.

Muscular Dystrophies

Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 1/5000 males and is the most common type of muscular dystrophy. DMD is caused by the absence of dystrophin (encoded by the gene DMD), which is a protein that links the sarcomere and the extracellular matrix by anchoring the sarcolemma to the outermost myofilament layer of myofibers. DMD is a progressive infantile neuromuscular condition that is marked by muscle wasting and weakness, skeletal deformations, loss of independent walking by the age of 10, respiratory dysfunction by the age of 20 and, ultimately, cardiopulmonary failure and death between ages 20 and 40 years. Despite increased awareness among clinicians, there is an average delay of 2.5 years between the onset of symptoms and the time of definitive diagnosis. Cardiovascular manifestations of DMD are most commonly represented by dilated cardiomyopathy (DCM), arrhythmias and congestive heart failure (HF). In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has DMD. In some embodiments, the method relates to treating a patient with HFrEF heart failure who has DMD.

The most frequent mutations found in patients with DMD (approximately 65%) are deletions of one or more exons of the dystrophin gene (DMD), which is one of the largest genes in the human genome, leading to the complete absence of the mature protein dystrophin. Duplications occur in 6%-10% of cases while nonsense, missense and deep intronic changes altogether account for the remaining 25% of molecular defects. Dystrophin is located on the inner side of the skeletal and cardiac sarcolemma and it interacts with a large number of membrane proteins, playing an important role in the regulation of signal transduction. The lack of dystrophin in a patient with DMD results in destabilization of the dystrophin-associated glycoprotein complex (DGC) causing sarcolemmal instability during the repeated cycles of contraction and relaxation, alongside with the reduction of the force transmission generated by sarcomeres. In the heart, along with membrane integrity, the lack of dystrophin affects L-type calcium channels and the function of the mechanical stretch-activated receptors. This results in increased levels of intracellular calcium, therefore, activating calpains and proteases that consequently degrade contractile proteins, promoting cellular death and fibrosis.

Current standards of care for DMD diagnosis suggest bypassing muscle biopsy and performing a genetic testing first. Since deletions and duplications of one or more exons are identified in the majority of patients, it is most cost-effective to check for these mutations first by multiplex ligation-dependent probe amplification (MLPA). The relevance of early genetic testing is evident in the fact that different types of mutations carry different prognostic and phenotypic characteristics. For example, deletions occurring at 5′-(exons 3-9) or 3′-ends (exons 48-52) of DMD are more often associated with heart involvement, although a mechanistic explanation is unclear.

It is recommended that baseline cardiac assessment be performed with ECG and echocardiography first at the age of 6 years, and then biannually until the age of 10 years in the absence of symptoms. A switch to regular annual assessments with ECG and echocardiography is recommended to be done at the age of 10 years or at the onset of cardiac signs and symptoms if they occur earlier. Sinus tachycardia is a common finding in patients with DMD during childhood, even when they are immobile, along with short PR interval and right ventricular hypertrophy. In some embodiments, a patient with heart failure and DMD is assessed using echocardiography. In some embodiments, a patient with heart failure and DMD is assessed using ECG.

Echocardiography and ECG are standardly used for screening and detection of cardiovascular abnormalities in DMD patients, although these tools are not always adequate to detect an early, clinically asymptomatic phases of disease progression. In this regard, cardiovascular magnetic resonance (CMR) with late gadolinium enhancement is emerging as a promising method for the detection of early cardiac involvement in patients with DMD. The early detection of cardiac dysfunction allows the therapeutic institution of various classes of drugs such as corticosteroids, beta-blockers, ACE inhibitors, antimineralocorticoid diuretics and novel pharmacological and surgical solutions in the multimodal and multidisciplinary care for this group of patients. In some embodiments, a patient with heart failure and DMD is assessed using CMR. In some embodiments, a patient with heart failure and DMD is assessed using CMR with late gadolinium enhancement.

There are several known treatments for heart failure that are known to be prescribed to DMD patients with HF. To delay the onset and/or treat LV dysfunction, corticosteroids (e.g., mineralcorticoids, glucocorticoids), inhibitors of the renin-angiotensin system (RAAS) (e.g., ACE inhibitors), and/or beta blockers are typically prescribed. Corticosteroids are the most relevant class of drugs introduced in the treatment of patients with DMD, having a profound impact on the natural history of the disease. Early steroid treatment effectively slows skeletal muscle wasting and dysfunction, preserves the ambulation and is associated with the reduction of scoliosis risk and pulmonary failure. Additionally, steroid administration may also have a positive impact on LV function in patients with DMD, although the exact mechanisms underlying this process are currently unclear. Furthermore, early treatment of patients with DMD aged from 9.5 to 13 years with one or more ACE inhibitors has been shown to delay the onset and progression of LV dysfunction. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more corticosteroids (e.g., mineralcorticoids, glucocorticoids), inhibitors of the renin-angiotensin system (RAAS) (e.g., ACE inhibitors), and/or beta blockers.

Furthermore, DMD patients may be prescribed one or more COX-inhibiting nitric oxide donors, which have been recently introduced in the treatment of patients with DMD. This class of drugs has a structure similar to non-steroidal anti-inflammatory drugs, but with higher capability of transporting nitric oxide, thus decreasing inflammation in both skeletal and cardiac muscles. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more COX-inhibiting nitric oxide donors.

Other therapies for treatment of DMD include, but are not limited to stop codon read-through approaches, viral vector-based gene therapy and antisense oligonucleotides (AON) for exon skipping (e.g., a morpholino). For gene therapy in DMD, a primary goal is to deliver a replacement copy of the dystrophin gene. To accomplish gene transfer in DMD, some aim to utilize the action of viruses, specifically AAV viruses, which will theoretically deliver the dystrophin gene into muscle cells to manufacture dystrophin protein. The large size of the dystrophin gene can pose a challenge because there is a limit to the size of the load that viruses can carry. To address this, a smaller, but still functional, version of dystrophin is typically used in gene therapy. Mini dystrophin (rAAV2.5-CMV-minidystrophin) is a miniaturized, working dystrophin gene that has been tested in boys with DMD. Moreover, an even smaller version of dystrophin called microdystrophin has been developed, which contains the minimum amount of information from the dystrophin gene needed to produce a functional protein. SGT-001 is a gene therapy that delivers engineered microdystrophin. Another similar drug is called rAAVrh74.MHCK7.micro-Dystrophin. Other gene therapies in development for treating DMD include, but are not limited to SRP-9001 and GALGT2. Encased in an adeno-associated virus (AAV) vector, or delivery vehicle, microdystrophin genes are administered systemically to the body via intravenous (IV) infusion. Antisense oligonucleotides (AON) for exon skipping are short, synthetic nucleic acid sequences which bind to complementary target mRNA sequences and lead to either endonuclease-mediated transcript knockdown or splice modulation. AON-mediated exon skipping can correct the reading frame by removing an out-of-frame exon or exons from the DMD pre-mRNA, producing a truncated but partly functional dystrophin protein. The first generation of AONs has an unmodified phosphoribose backbone, making them susceptible to degradation by nucleases. Second and third generation AONs contain chemically modified structures that not only increase AON resistance to nuclease degradation, but also enhances their pharmacological properties. Phosphorodiamidate morpholino oligomers (PMOs) represent the most advanced use of antisense therapy for DMD. PMOs have the deoxyribose/ribose moiety replaced by a morpholine ring, and the charged phosphodiester inter-subunit linkage replaced by an uncharged phosphorodiamidate linkage, making PMOs nuclease-resistant and charge-neutral, which imparts an even greater resistance to nucleases that typically target charged molecules. Additionally, due to the lack of charge, PMOs are safer since they are unlikely to activate Toll-like receptors, a class of receptors involved in producing innate immune responses against pathogenic material. RNAi technologies for treating DMD include, but are not limited to, eteplirsen (SRP-4051), golodirsen (SRP-4053), casimersen (SRP-4045), peptide-conjugated eteplirsen (SRP-5051), SRP-5053, SRP-5045, SRP-5052, SRP-5044, SRP-5050, viltolarsen (NS-065/NCNP-01), NS-089/NCNP-02 (exon skipping 44), DS-5141b (exon skipping 45), suvodirsen (WVE-210,201), and drisapersen (PRO051). Other similar therapies include single-stranded oligodeoxynucleotides (ssODNs) made of peptide nucleic acids (PNA) (e.g., a PNA-ssODN targeting DMD exon 10), a chimeric peptide-PMO conjugate (e.g., a conjugating a muscle-specific peptide (MSP) and a cell-penetrating peptide (B peptide) with a phosphorodiamidate morpholino oligomer (PMO), M12-PMO), M12-PMO (exon 23 skipping), and M12-PMO (exon 10 skipping). Aminoglycoside-derived compounds have been recently used in patients with DMD since they bind the 60S subunit of ribosomes and ‘relax’ the premature stop codons, with no evident effects on the naïve nonsense triplets. A compound belonging to this class, Atalurenhas, has been recently approved for the treatment of DMD. Overexpression of utrophin, a protein very similar to dystrophin, has been proven as a partial rescuer of dystrophin expression. Several other therapeutic strategies aim to target the disease progression by reducing or preventing muscle necrosis and fibrosis (e.g., tadalafil) or by increasing muscle mass (e.g., myostatin inhibitors) are currently under investigation. Finally, cell therapy enables transplantation of normal dystrophin-expressing satellite cells into patient's skeletal muscle, in order to obtain a fusion with resident muscle fibers and consequently the spreading of dystrophin expression to patient's cells. Although not tested on cardiomyocytes, these approaches are potentially promising for the restoration of dystrophin in the heart. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more stop codon read-through approaches, viral vector-based gene therapies, antisense oligonucleotides (AON) for exon skipping, Atalurenhas, utrophin overexpression, tadalafil, myostatin inhibitors, and cell therapies. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more of rAAV2.5-CMV-minidystrophin, SGT-001, rAAVrh74.MHCK7.micro-Dystrophin, SRP-9001, and GALGT2. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and rAAV2.5-CMV-minidystrophin. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SGT-001. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and rAAVrh74.MHCK7.micro-Dystrophin. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SRP-9001. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and GALGT2. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more of eteplirsen (SRP-4051), golodirsen (SRP-4053), casimersen (SRP-4045), peptide-conjugated eteplirsen (SRP-5051), SRP-5053, SRP-5045, SRP-5052, SRP-5044, SRP-5050, viltolarsen (NS-065/NCNP-01), NS-089/NCNP-02 (exon skipping 44), DS-5141b (exon skipping 45), suvodirsen (WVE-210,201), drisapersen (PRO051), PNA-ssODN, M12-PMO (exon 23 skipping), and M12-PMO (exon 10 skipping). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and eteplirsen. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and golodirsen. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and casimersen (SRP-4045). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and peptide-conjugated eteplirsen (SRP-5051). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SRP-5053. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SRP-5045. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SRP-5052. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SRP-5044. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and SRP-5050. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and viltolarsen. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and NS-089/NCNP-02 (exon skipping 44). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and DS-5141b (exon skipping 45). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and suvodirsen (WVE-210,201). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and drisapersen. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and PNA-ssODN. In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and M12-PMO (exon 23 skipping). In some embodiments, the method relates to treating heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and M12-PMO (exon 10 skipping).

Limb Girdle Muscular Dystrophy

Limb Girdle muscular dystrophy (LGMD) refers to a large collection of progressive muscle diseases with proximal weakness greater than distal weakness. It is characterized by progressive muscle wasting which affects predominantly hip and shoulder muscles. Autosomal dominant conditions are designated as LGMD 1X, where X currently ranges from A to H, and autosomal recessive conditions are LGMD 2X, where X currently ranges from A to Q. The list of LGMDs is long and continues to expand, with new letters in each category appearing on a regular basis. Some of the more common LGMDs are types 1A, 1B, 1C, 2A, 2B, 2C-2F, 2I, and 2L. LGMD type 1A involves one or more mutations in the myotilin (MYOT) gene. LGMD type 1B involves one or more mutations in the lamin A/C (LAMNA) gene. LGMD type 1C involves one or more mutations in the Caveolin-3 (CA V3) gene. LGMD type 2A involves one or more mutations in the Calpain-3 (CAPN3) gene. LGMD type 2B involves one or more mutations in the Dysferlin (DYSF) gene. LGMD types 2C-F involve one or more mutations in the γ-Sarcoglycan (SGCG), α-Sarcoglycan (SGCA), β-Sarcoglycan (SGCB), and/or δ-Sarcoglycan (SGCD) genes, respectively. LGMD type 21 involves one or more mutations in the FKRP gene. LGMD type 2L involves one or more mutations in the Anoctamin-5 (ANO5) gene.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient also has limb girdle muscular dystrophy. In some embodiments, the method relates to treating a patient with HFrEF heart failure who has limb girdle muscular dystrophy. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the myotilin (MYOT) gene. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the lamin A/C (LMNA) gene. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the Caveolin-3 (CAV3) gene. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the Calpain-3 (CAPN3) gene. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the Dysferlin (DYSF) gene. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the γ-Sarcoglycan (SGCG), α-Sarcoglycan (SGCA), β-Sarcoglycan (SGCB), and/or δ-Sarcoglycan (SGCD) genes. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the fukutin-related protein (FKRP) gene. In some embodiments, a patient with limb girdle muscular dystrophy and heart failure has one or more mutations in the Anoctamin-5 (ANO5) gene.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more of SRP-9003, SRP-9004, SRP-9005, SRP-6004, SRP-9006, and LGMD2A.

Friedreich's Ataxia

Friedreich's ataxia (FRDA or FA) is an autosomal recessive genetic disease that causes difficulty walking, a loss of sensation in the arms and legs and impaired speech that worsens over time. Symptoms can start between 5 and 15 years of age, and many patients develop hypertrophic cardiomyopathy and will require a mobility aid such as a cane, walker or wheelchair in their teenage years. As the disease progresses, patients lose their sight and hearing. Other complications include scoliosis and diabetes mellitus. In the heart FRDA patients often develop sorne fibrosis, and over time many patients develop left ventricle hypertrophy and dilatation of the left ventricle.

The condition is caused by mutations in the FXN gene on chromosome 9. The FXN gene encodes a protein called frataxin. In FRDA, a patient produces less frataxin. Degeneration of nerve tissue in the spinal cord causes the ataxia; particularly affected are the sensory neurons essential for directing muscle movement of the arms and legs through connections with the cerebellum. The spinal cord becomes thinner and nerve cells lose some myelin sheath.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient also has Friedrich's ataxia. In some embodiments, the method relates to treating a patient with HFrEF heart failure who has Friedreich's ataxia muscular dystrophy. In some embodiments, a patient with Friedreich's ataxia muscular dystrophy and heart failure has one or more mutations in the frataxin (FAN) gene.

Myotonic Dystrophy

Myotonic dystrophy is a long-term autosomal dominant genetic disorder that affects muscle function. Symptoms include gradually worsening muscle loss and weakness, and muscles often contract and are unable to relax (myotonia). Other symptoms may include cataracts, intellectual disability and heart conduction problems. Myotonic dystrophy affects more than 1 in 8,000 people worldwide. While myotonic dystrophy can occur at any age, onset is typically in the 20s and 30s.

There are two main types of Myotonic dystrophy: type 1, due to mutations in the DMPK gene which encodes for myotonic dystrophy protein kinase, and type 2, due to mutations in the CNBP gene, which encodes for CCHC-type zinc finger nucleic acid binding protein. Presentation of symptoms and signs varies considerably by type, with type 2 tending to be a more mild disease. Symptoms may appear at any time from infancy to adulthood. Myotonic dystrophy causes general weakness, usually beginning in the muscles of the hands, feet, neck, or face. It slowly progresses to involve other muscle groups, including the heart. Muscle weakness associated with type 1 particularly affects the lower legs, hands, neck, and face. Muscle weakness in type 2 primarily involves the muscles of the neck, shoulders, elbows, and hips.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient also has Myotonic dystrophy. In some embodiments, the method relates to treating a patient with HFrEF heart failure who has Myotonic dystrophy. In some embodiments, a patient with Myotonic dystrophy and heart failure has one or more mutations in the myotonic dystrophy protein kinase (DMPK) gene. In some embodiments, a patient with Myotonic dystrophy and heart failure has one or more mutations in the CCHC-type zinc finger nucleic acid binding protein (CNBP) gene.

Dilated Cardiomyopathy (DCM)

Dilated cardiomyopathy (DCM) is the second most common etiology of HF with reduced ejection fraction (HFrEF). It is a heterogeneous disorder with multiple etiologies of its own, though it is estimated that 20 to 50% of DCM is caused by a genetic mutation inherited in a Mendelian fashion. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of dilated cardiomyopathy comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to treating a patient with HFrEF who has DCM.

DCM is characterized pathologically by dilatation of the left ventricle, functionally by progressive contractile failure, and histologically by cardiomyocyte hypertrophy, loss of myofibrils, and interstitial fibrosis. Patients with DCM may be initially asymptomatic but develop exertional dyspnea, orthopnea, and fatigue as the left ventricle fails. Right ventricular failure is frequently present because of concurrent involvement by cardiomyopathy or secondary to left ventricular failure. Complications of DCM such as arrhythmia, mitral regurgitation, or embolization of intracardiac thrombus may be presenting features of the disease. Mortality is significant through progressive HF or arrhythmic sudden death. In some embodiments, the disclosure relates to a method of treating a patient having dilation of the left ventricle. DCM itself can be caused by diverse insults to the heart, one of which being genetic disease. In some embodiments, the disclosure relates to treating a patient having progressive contractile failure. In some embodiments, the disclosure relates to a method of treating a patient having one or more of cardiomyocyte hypertrophy, loss of myofibrils, and interstitial fibrosis. In some embodiments, the disclosure relates a method of treating a patient having a genetic form of DCM.

There are over 50 currently recognized genes associated with DCM, most of which encode proteins in the cardiomyocyte sarcomere. Key disease genes in DCM are shown in Table 7. Mutations currently have been identified in approximately 30% to 35% of patients with familial DCM, with the following 4 genes accounting for the majority: titin (TTN), lamin A/C (LMNA), β-myosin heavy chain (MYH7), and cardiac troponin T (TNNT2). Titin mutations appear to be the most common. If conduction abnormalities are present, then a mutation in LMNA is typically found in up to one third of cases. The multiplicity of genes reported represents diverse cellular pathways, all of which converge on a macroscopic DCM phenotype that is not obviously clinically distinguishable. Although there does not appear to be a unifying cellular pathophysiology, DCM genes can be broadly grouped by pathogenetic effect on contractile force generation and regulation, force transduction and mechanosensing, and nuclear proteins and transcription factors (Table 7). Beyond these categories, further candidate genes with widespread cellular effects have been proposed and will continue to emerge, for example, on ion channel function, autophagy, and mitochondrial regulation, but remain to be fully validated or replicated as mechanistic pathways leading to DCM.

TABLE 7
Key Disease Genes in Dilated Cardiomyopathy (DCM)
Gene   Description
Force generation/regulation
ACTC1  Actin, α, cardiac muscle 1
MYH7   Myosin, heavy chain 7, cardiac muscle, β
TNNT2  Troponin T type 2 (cardiac)
TNNI3  Troponin I type 3 (cardiac)
TNNC1  Troponin C type 1 (slow)
PLN    Phospholamban
Force transduction/mechanosensing
TTN    Titin
DMD    Dystrophin
SGCD   Sarcoglycan, Δ (35-kDa dystrophin-associated)
DES    Desmin
Nuclear proteins/transcription
LMNA   Lamin A/C
Other
RBM20  RNA-binding motif protein 20
Implicated but not yet supported by linkage/cosegregation
ACTN2  Actinin, α 2
VCL    Vinculin
TMPO   Thy mopoetin
TCAP   Titin-cap
BAG3   BCL2-associated athanogene 3
LDB3   LIM domain-binding 3
ANKRD1 Ankyrin repeat domain 1 (cardiac muscle)

Mutations in proteins of both the thick and thin myofilaments can cause DCM. β-Myosin heavy chain (MYH7), intrinsic to contractile force generation by type II myosin, is the sarcomeric gene most commonly mutated in DCM. MYH7 mutations are found in 5% to 10% of DCM cases. Allelic heterogeneity is extensive, with numerous different mutations reported. The thin-filament protein cardiac actin (ACTC1) is a rare cause of DCM, in addition to HCM and LVNC, but was the first sarcomeric DCM gene identified. Mutations at the ACTC1 dystrophin-binding site are typically associated with the development of DCM. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in MYH7. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in ACTC1. In some embodiments, the disclosure relates to a method of treating a patient with DCM that has one or more mutations in ACTC1 and MYH7.

Regulation of sarcomeric contraction is mediated primarily by tropomyosin and the troponin complex, composed of T, I, and C subunits. Troponin T (TNNT2), which binds tropomyosin, regulates interaction of the troponin complex with the thin filament. Troponin I (TNNI3) modulates sarcomeric activation through an inhibitory effect on actin-myosin binding during diastole, and troponin C (TNNC1) binds calcium during systole and promotes cross-bridge formation between actin and myosin, leading to contraction. DCM-causing mutations have been identified in all 3 subunits and are associated with impaired calcium sensitivity. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in TNNT2. In some embodiments, the disclosure relates to a method of treating a patient with DCM has one or more mutations in TNNI3. In some embodiments, the disclosure relates to a method of treating a patient with DCM has one or more mutations in TNNC1.

Beyond the sarcomere, dysregulation of contractile force generation is clearly linked to development of DCM. Phospholamban (PLN) is a small, highly conserved phosphoprotein that modulates sarcoplasmic reticulum calcium uptake, influencing downstream force production by the myofilament. Mutations in PLN cause autosomal-dominant DCM and are rare but well characterized, with the Arg14del mutation notable for its association with ventricular arrhythmia. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in PLN.

Efficient transmission of force across the sarcomere, cell cytoskeleton, and extracellular matrix is essential to normal cardiac contractile function. Multiple proteins with a primary structural function or those that act as mechanosensors that modulate the sarcomere are linked to familial DCM. Key examples are titin, dystrophin, and desmin. The giant protein titin, spanning half the sarcomere from the Z disk to the M line, functions as an elastic molecular spring regulating passive tension and active contraction. The titin gene (TTN) contains 363 exons and approximately 33,000 amino acids and interacts with >20 other structural, signaling, and modulatory proteins, including telethonin, α-actinin, and possibly muscle LIM in a putative mechanosensor complex at the Z disk. The spectrum of titinopathies also includes skeletal muscle phenotypes, including limb-girdle muscular dystrophy, tibial muscular dystrophy, and hereditary myopathy with early respiratory failure; however, there is little evidence as yet of skeletal muscle involvement with the DCM-causing mutations. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in TTN.

Another example of a key protein in force transduction is dystrophin (DMD), the first DCM disease gene reported. In addition to X-linked dilated cardiomyopathy (DCM), mutations in DMD cause Duchenne and Becker muscular dystrophies which are characterized by progressive skeletal muscle weakness. Dystrophin is a large cytoskeletal protein which forms a transmembrane link between the sarcomere and the extracellular matrix, the dystrophin-associated glycoprotein complex, alongside other proteins such as the sarcoglycans. Mutations in δ-sarcoglycan (SGCD) also have been implicated in DCM, although typically they cause limb-girdle muscular dystrophy. Desmin (DES) mutations are a rare cause of DCM but are significant for an association with arrhythmias alongside HF. A founder mutation in the desmin single-head domain has been reported to cause a predominantly right ventricular cardiomyopathy with conduction disease. Desmin is an intermediate filament protein that, with microfilaments and microtubules, maintains the cytoskeletal infrastructure and subcellular spatial organization. In addition to DCM, desmin mutations can cause skeletal muscle disease, including myofibrillar myopathy and scapuloperoneal syndrome. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in DMD. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in DES. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in SGCD.

DCM caused by LMNA mutations is clinically distinctive because it is associated with progressive conduction disease, initially atrioventricular block, and high risk of sudden cardiac death (SCD). Conduction abnormalities typically precede the development of DCM, which may be isolated or involve associated skeletal muscle disease. Other rare cardiac phenotypes also have been reported, including early atrial fibrillation, LVNC, RCM, and HCM. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in LMNA.

Other genes implicated in DCM are indicated in Table 7. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in RBM20. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in ACTN2. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in VCL. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in TMPO. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in TCAP. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in BAG3. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in LDB3. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more mutations in ANKRDT.

Development of genetic DCM occurs over time, normally with a prolonged asymptomatic phase during which the heart is initially macroscopically normal both morphologically and functionally. When an individual is known to be genotype positive and phenotype negative (e.g., an individual in a family with known DCM), the timing and severity of a phenotype are difficult to predict, given the variability of expressivity and penetrance. With serial follow-up, imaging by echocardiography or magnetic resonance imaging will usually detect abnormalities of cardiac size or function before overt symptoms. Once clear symptoms develop, there is an approximate correlation with the degree of left ventricular dysfunction, although other factors such as diastolic function, arrhythmia, mitral regurgitation, right side HF, and other comorbidities will interact.

Given its prevalence in the general population, a family history of HF alone is not usually sufficient to indicate a diagnosis of familial DCM, though there are some strongly suggestive features (e.g., heart attack in a 1^st degree relative younger than 55 years old (male) or 65 years old (female), sudden unexplained death, recurrent or unexplained syncope or near syncope, heart failure in young, 1^st degree relative younger than 60 years old, or heart transplantation in a 1^st degree relative). The pattern of inheritance in DCM is most frequently autosomal dominant, though penetrance is reduced such that all not all family members who harbor a mutation will develop DCM. There are X-linked, autosomal recessive and mitochondrial forms of heritable DCM as well. Expressivity also is variable, and the severity of the DCM phenotype may vary widely among affected family members. In some embodiments, the disclosure relates to a method of treating a patient with DCM having autosomal dominant DCM. In some embodiments, the disclosure relates to a method of treating a patient with DCM having autosomal recessive DCM. In some embodiments, the disclosure relates to a method of treating a patient with DCM having X-linked DCM. In some embodiments, the disclosure relates to a method of treating a patient with DCM having mitochondrial DCM.

Approximately 25% of patients with dilated cardiomyopathy (DCM) will have evidence of mid-wall fibrosis which is an independent predictor of mortality and morbidity. DCM patients with mid-wall fibrosis had a similar outcome to those with ischemic disease. Thus, as with ischemic cardiomyopathy, the presence of fibrosis/scar is a marker of adverse outcome and worse response to device therapy.

DCM is a left ventricular dilated phenotype of heart failure. Dilated phenotypes are a heterogenous group characterized by large left ventricular (LV) cavities with eccentric remodeling or hypertrophy and impaired contractility. Such phenotypes can be a response to abnormal loading conditions typically in valvular disease or hypertension, severe coronary or congenital disease, or predominantly confined to heart muscle like in inherited or acquired cardiomyopathies such as DCM. Transthoracic echocardiography is used as a first line imaging tool for identifying a phenotype. Images typically show global left or biventricular hypokinesis with or without regional wall motion abnormalities. Ventricular and atrial dilatation, intracardiac thrombi and functional mitral regurgitation due to annular dilatation might also be noted. Doppler parameters can assist in quantifying valvular abnormalities and the severity of diastolic dysfunction. In some embodiments, the disclosure relates to a method of treating a patient with DCM having one or more of large left ventricular (LV) cavities with eccentric remodeling or hypertrophy and impaired contractility.

While echocardiography is a first-line diagnostic tool for DCM, volumes and ejection fraction (EF) acquired from 3D echocardiography correlate better with cardiac magnetic resonance imaging (CMR) and its use is recommended when feasible. CMR plays a central role in phenotypic assessment.

Hypertrophic Cardiomyopathy (HCM)

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease, with a prevalence of approximately 1 in 500. HCM is characterized by inappropriate myocardial hypertrophy, which develops in the absence of pressure overload (e.g., hypertension, aortic stenosis) or infiltration (e.g., amyloidosis). Hypertrophy in HCM classically affects the interventricular septum, causing left ventricular outflow tract obstruction, but may be apical, segmental, or concentric. The histological disease features are interstitial fibrosis, myocyte enlargement, and disarray. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of hypertrophic cardiomyopathy (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), In some embodiments, the method relates to treating a patient with HFrEF who has HCM. In some embodiments, the method relates to treating a patient with HCM having inappropriate myocardial hypertrophy in the absence of pressure overload and/or infiltration. In some embodiments, the method relates to treating a patient with HCM having one or more of interstitial fibrosis, myocyte enlargement, and/or disarray.

Up to 20% of patients with HCM develop HF at a median age of 48±19 years, and rates of HF are likely to increase as mortality from sudden cardiac death reduces with ICD implantation. Three HF subtypes are clinically described. First, about 30% of HCM patients with HF have development of progressive left ventricular dilatation, thinning, and systolic dysfunction, described as “burnt-out” HCM. Approximately 20% of HCM patients with HF have development of left ventricular systolic dysfunction attributable to pressure overload by left ventricular outflow tract obstruction. Finally, up to 50% of HCM patients with HF show evidence of diastolic HF, with a normal or supernormal ejection fraction but impaired ventricular relaxation, elevated end-diastolic pressure, left atrial enlargement, and atrial fibrillation. In some embodiments, the disclosure relates to a method of treating a patient having progressive left ventricular dilatation, thinning, and systolic dysfunction. In some embodiments, the disclosure relates to a method of treating a patient having “burnt-out” HCM. In some embodiments, the disclosure relates to a method of treating a patient having left ventricular systolic dysfunction attributable to pressure overload by left ventricular outflow tract obstruction. In some embodiments, the disclosure relates to a method of treating a patient having diastolic HF, with a normal or supernormal ejection fraction but impaired ventricular relaxation, elevated end-diastolic pressure, left atrial enlargement, and atrial fibrillation.

HCM is primarily a disease of the sarcomere, with mutations in eight sarcomere genes (Table 8) encoding contractile or regulatory proteins detected in approximately 60% of clinical cohorts. At a cellular level, HCM mutations lead to increased myofilament sensitivity and affinity to calcium and increased actin-activated ATPase activity. Like DCM and AC, inheritance is typically autosomal dominant, with locus and allelic heterogeneity, and there is usually a silent compensatory period before emergence of a variable phenotype. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in a sarcomere gene. In some embodiments, a patient has an autosomal dominant mutation.

The most common HCM genes, β-myosin heavy chain (MYH7) and myosin-binding protein C (MYBPC3), together account for approximately 50% of HCM disease. Approximately 200 mutations in MYH7 alone have been found. Other key genes are shown in Table 8. The remaining sarcomeric genes are cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), α-tropomyosin (TPM1), cardiac actin (ACTC1), essential myosin light chain 3 (MYL3), and regulatory myosin light chain (MYL2). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in β-myosin heavy chain (MYH7).

In some embodiments, the disclosure relates to a method of treating a patient having a mutation in myosin-binding protein C (MYBPC3). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in cardiac troponin T (TNNT2). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in cardiac troponin I (TNNI3). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in α-tropomyosin (TPM1). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in cardiac actin (ACTC1). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in essential myosin light chain 3 (MYL3). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in regulatory myosin light chain (MYL2).

In about 40% of HCM patients, no causative mutation can be identified in known HCM disease genes (Table 8). This may imply that further genes remain to be defined, or that nonmendelian inheritance or nongenetic factors also play a role. The model of HCM as a monogenic disease following mendelian patterns of inheritance is increasingly recognized as an oversimplification. Beyond pathogenic mutations, genetic, epigenetic, and environmental modifiers of the HCM phenotype are important but not yet well understood. These factors underlie the great phenotypic variability, in both the pattern of hypertrophy and the clinical course, in patients with the same genotype.

TABLE 8
Key Disease Genes in Hypertrophic Cardiomyopathy (HCM)
Gene   Description
Sarcomeric
MYH7   Myosin, heavy chain 7, cardiac muscle, β
MYBPC3 Myosin-binding protein C, cardiac
TNNT2  Troponin T type 2 (cardiac)
TNNI3  Troponin I type 3 (cardiac)
TPM1   Tropomyosin 1 (a)
MYL2   Myosin, light chain 2, regulatory cardiac, slow
MYL3   Myosin, light chain 3, alkali, ventricular, skeletal, slow
ACTC1  Actin, a, cardiac muscle 1
Non-sarcomeric
CSRP3  Cysteine and glycine-rich protein 3 (cardiac LIM protein)
Hypertrophic cardiomyopathy phenocopies
PRKAG2 Protein kinase, AMP-activated, y 2 noncatalytic subunit
LAMP2  Lysosomal-associated membrane protein 2
GLA    Galactosidase, α
FHL1   Four-and-a-half LIM domains 1

Arrhythmogenic Cardiomyopathy (AC)

Arrhythmogenic Cardiomyopathy (AC) is characterized by progressive fibrofatty replacement of the ventricular myocardium, leading to arrhythmia, HF, and SCD in patients. It is classically described as a disease of the right ventricle, sometimes referred to as arrhythmogenic right ventricular cardiomyopathy (ARVC), but left ventricular involvement is increasingly recognized. Left ventricular AC is distinguished from DCM by its patchy involvement and a disproportionate propensity to arrhythmia for a given degree of systolic dysfunction. Because there may be right, left, or biventricular involvement, the phenotype has been more accurately renamed AC. Characteristic histological findings are patchy fibrosis, inflammation, myocyte death, wall thinning, and aneurysm formation. AC classically presents in a proband with malignant arrhythmia, which may cause sudden cardiac death as the first manifestation of disease in adolescence or young adulthood. A “concealed” phase, with arrhythmic features, typically precedes overt cardiomyopathy. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of arrhythmogenic cardiomyopathy comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to treating a patient with HFrEF who has AC. In some embodiments, the method relates to treating a patient with AC with progressive fibrofatty replacement of the ventricular myocardium.

In some embodiments, the disclosure relates to a method of treating a patient having progressive fibrofatty replacement of the ventricular myocardium. In some embodiments, the disclosure relates to a method of treating a patient having arrhythmia. In some embodiments, the disclosure relates to a method of treating a patient having one or more of patchy fibrosis, inflammation, myocyte death, wall thinning, and aneurysm formation.

Between about 10% and about 20% of patients with AC will develop HF, with right or left (or both) ventricular systolic dysfunction, which may rarely be the presenting feature of the disease. AC is a familial disease in greater than 50% of cases, with an estimated prevalence of 1 in 1000 to 1 in 5000. Like DCM and HCM, AC is heterogeneous in phenotype, genotype, and allele. It is classically described as autosomal-dominantly inherited, but this is likely to be an oversimplification, with many patients carrying mutations in >1 disease gene (double or compound heterozygosity). Penetrance, which is age dependent as in other cardiomyopathies, is low; Two autosomal-recessive forms of AC have been described—the cardiocutaneous disorders Naxos disease and Carvajal syndrome—that comprise AC, palmoplantar keratoderma, and woolly hair. In some embodiments, the disclosure relates to a method of treating a patient having right ventricular systolic dysfunction. In some embodiments, the disclosure relates to a method of treating a patient having left ventricular systolic dysfunction. In some embodiments, the disclosure relates to a method of treating a patient having right and left ventricular systolic dysfunction. In some embodiments, the disclosure relates to a method of treating a patient having an autosomal dominant mutation. In some embodiments, the disclosure relates to a method of treating a patient having Naxos disease. In some embodiments, the disclosure relates to a method of treating a patient having Carvajal syndrome.

AC has emerged genetically as a “disease of the desmosome,” with pathogenic mutations identified in 5 genes encoding the desmosomal complex (Table 9). Desmosomes are symmetrical linkage complexes that span the intercellular membrane and fulfill strengthening and signaling roles, contributing to the intercalated disk. They consist of desmosomal cadherins (e.g., desmocollin 2 (DSC2) and desmoglein 2 (DSG2)), armadillo proteins (including junctional plakoglobin (JUP) and plakophilin 2 (PKP2)), and plakins (e.g., desmoplakin (DSP)). DSG2 and DSC2 form the transmembrane component of the desmosome and are anchored within the cell by plakoglobin and plakophilin 2, which bind the N-terminal domain of desmoplakin. Desmoplakin is, in turn, linked to desmin intermediate filaments at its C-terminal. In additional to structural roles, the desmosome is linked to the Wnt/β-catenin signaling pathway by plakophilin 2, which translocates to the nucleus to modify gene expression. Pathogenic mutations in AC cause mislocalization and reduction in desmosome number, remodeling of the intercalated disk with associated abnormal formation of gap junctions, and misincorporation of desmoplakin/plakoglobin. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in desmocollin 2 (DSC2). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in desmoglein 2 (DSG2). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in desmoplakin (DSP). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in junctional plakoglobin (JUP). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in plakophilin 2 (PKP2). In some embodiments, the disclosure relates to a method of treating a patient having a mutation in transmembrane protein 43 (TMEM43).

Several extradesmosomal genes are reported to cause AC, including TMEM43 and TGFB3. Further candidate genes for AC also have been proposed, including TTN and PLN, although these are not supported by linkage, and there is blurring of phenotypic boundaries between classic arrhythmogenic right ventricular cardiomyopathy, left-dominant AC, and DCM with arrhythmia. Mutations in LMNA recently have been reported to mimic the AC phenotype.

TABLE 9
Key Disease Genes in Arrhythmogenic Cardiomyopathy (AC)
Gene   Description
Demosomal
PKP2   Plakophilin 2
DSC2   Desmocollin 2
DSG2   Desmoglein 2
DSP    Desmoplakin
JUP    Junction plakoglobin
Extradesmosomal
TMEM43 Transmembrane protein 43

Left Ventricular Noncompaction Cardiomyopathy (LVNC)

Left ventricular noncompaction cardiomyopathy (LVNC) is an uncommon but increasingly recognized cardiomyopathy, either sporadic or familial, in which deep trabeculation of the myocardium is associated with progressive contractile dysfunction. The LVNC phenotype overlaps extensively with HCM and DCM and frequently occurs alongside structural heart disease, for example, Ebstein anomaly, pulmonary atresia, atrial/ventricular septal defects, and patent ductus arteriosus. It is also a feature of multisystem disorders involving the heart, including Barth and Noonan syndromes. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of left ventricular noncompaction cardiomyopathy comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to treating a patient with HFrEF who has LVNC. In some embodiments, the method relates to treating a patient with HFrEF who has familial LVNC. In some embodiments, the method relates to treating a patient with HFrEF who has sporadic LVNC. In some embodiments, the method relates to treating a patient with LVNC, wherein deep trabeculation of the myocardium is associated with progressive contractile dysfunction.

The pathognomonic feature of LVNC is a noncompacted, 2-layer myocardium. Persisting noncompaction from the embryological developing heart has been proposed to underlie the pathogenesis, although a normal myocardial appearance before the development of LVNC has been reported. Diagnostic sensitivity for LVNC is significantly higher with cardiac magnetic resonance imaging than with echocardiography.

LVNC manifests clinically with HF, thromboembolism, arrhythmia, or sudden cardiac death. Systolic dysfunction and diastolic dysfunction are common, with HF reported at presentation in over half of the patients. The precise mechanism behind the development of HF is unclear, but microvascular ischemia and fibrosis are both likely contributory factors. Presentation may occur in utero, in infancy, in childhood, or in adulthood, and this varies extensively even within families.

TAZ was the first gene implicated in LVNC, in patients with Barth syndrome, an X-linked disease causing DCM or LVNC, skeletal myopathy, cyclic neutropenia, and growth restriction. TAZ encodes a family of proteins called the tafazzins, which have an acyltransferase function necessary for remodeling of mitochondrial cardiolipin, in turn required for normal mitochondrial morphology and OXPHOS. When an LVNC phenotype is seen consistently in family members (as opposed to occurring in individuals in a family with otherwise typical HCM or DCM), mutations in sarcomeric, cytoskeletal, and nuclear membrane genes have been found (e.g., mutations in MYH7, ACTC, TNNT2, MYBPC3, and TPM1). Inheritance may be autosomal dominant, recessive, or X-linked, and penetrance is variable. Even in defined cohorts with LVNC, the yield of mutations from screening known disease genes remains low. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in TAZ. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in MYH7. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in ACTC. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in TNNT2. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in MYBPC3. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in TPM1.

LVNC phenotypes also have been reported along with congenital heart defects, primarily ventricular septal defect, caused by mutations in α-dystrobrevin (DTNA). α-Dystrobrevin contributes to the dystrophin-associated glycoprotein complex, which is required for normal linkage of the extracellular matrix to the dystrophin-based cytoskeleton. Mutations in α-dystrobrevin also cause a muscular dystrophy phenotype. In some embodiments, a patient has a mutation in DTNA.

In addition to DCM, mutations in LDB3 (Cypher/ZASP) are reported in LVNC. There are reports of sporadic or familial disease associated with mutations in several other genes, including LMNA, MIB1, mitochondrial genes, and chromosomal imbalance/deletions. Variants in SCN5a have been proposed to modify arrhythmia risk. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in LDB3. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in LMNA. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in MIB1.

Restrictive Cardiomyopathy (RCM)

Restrictive Cardiomyopathy (RCM) is a rare cardiomyopathy characterized by impaired ventricular filling and diastolic function with relatively normal ventricular wall thickness and systolic function. The etiology of RCM is broad, including genetic disease (sporadic or familial), infiltration (e.g., amyloidisis, sarcoidosis), connective tissue disease (e.g., systemic sclerosis), glycogen storage disease, drugs, and radiation. A proportion remains idiopathic, which is likely to be genetic, with no clear causative mutation known. Restrictive physiology is a feature of several other cardiomyopathies, particularly HCM, and there is some overlap in these 2 phenotypes. Quite commonly, individuals with classic RCM features are identified in families in which most affected members have typical HCM. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of restrictive cardiomyopathy comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to treating a patient with HFrEF who has RC. In some embodiments, the method relates to treating a patient with HFrEF who has RC with impaired ventricular filling and diastolic function with relatively normal ventricular wall thickness and systolic function.

The prognosis of RCM, particularly in children, is poor, with worse outcomes than either HCM or DCM and with 5-year transplantation-free survival of only about 22%. Elevated end-diastolic left ventricular pressure leading to atrial enlargement, atrial fibrillation, and risk of thromboembolism is common. There is progression from diastolic dysfunction to refractory systolic HF, frequently necessitating heart transplantation.

Some patients with RCM were found to carry mutations in TNNI3. Mutations in several sarcomeric genes have subsequently been reported in patients with RCM, but in the majority of cases, there is no convincing association between the allele reported and a specific phenotype of RCM. Nonsarcomeric RCM mutations also have been reported. In addition to DCM, mutations in the intermediate filament protein desmin (DES) can cause an RCM phenotype with conduction disease. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in TNNI3. In some embodiments, the disclosure relates to a method of treating a patient having a mutation in DES.

7. Diagnosis of Heart Failure

Diagnosis of HFpEF remains challenging. In an HFpEF patient, LVEF is normal and signs and symptoms for HF are often non-specific and do not discriminate well between HF and other clinical conditions. Diagnosis of chronic HFpEF, especially in a typical elderly patient with co-morbidities and no obvious signs of central fluid overload, is cumbersome and a validated gold standard is elusive. To improve the specificity of diagnosing HFpEF, a clinical diagnosis should be supported by objective measures of cardiac dysfunction at rest or during exercise. A diagnosis of HFpEF typically requires the following: presence of symptoms and/or signs of HF; a ‘preserved’ EF (defined as LVEF≥50% or sometimes as 40-49% for HFmrEF); elevated levels of NPs (BNP>35 pg/mL and/or NT-proBNP>125 pg/mL); objective evidence of other cardiac functional and structural alterations underlying HF; and in case of uncertainty, a stress test or invasively measured elevated LV filling pressure may be needed to confirm the diagnosis.

Natriuretic Peptides

Plasma concentration of natriuretic peptides (NPs), including BNP and NT-proBNP, can be used as an initial diagnostic test, especially in a non-acute setting when echocardiography is not immediately available. Elevated NPs help establish an initial working diagnosis, identifying those who require further cardiac investigation. Patients with values below the cutoff point for the exclusion of important cardiac dysfunction typically do not require echocardiography. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has elevated level of one or more natriuretic peptides. In some embodiments, the method relates to treating a patient having heart failure wherein the patient has elevated levels of BNP. In some embodiments, the method relates to treating a patient having heart failure wherein the patient has elevated levels of NT-proBNP. In some embodiments, the patients NP (e.g., BNP and/or NT-proBNP) is elevated compared to a healthy people of similar age and sex.

Both BNP and NT-proBNP are markers of atrial and ventricular distension due to increased intracardiac pressure. The New York Heart Association (NYHA) developed a 4-stage functional classification system for congestive heart failure (CHF) based on the severity of symptoms. Studies have demonstrated that the measured concentrations of circulating BNP and NT-proBNP increase with the severity of CHF based on the NYHA classification.

Patients with normal plasma NP concentrations are unlikely to have HF. The upper limit of normal in the non-acute setting for B-type natriuretic peptide (BNP) is 35 pg/mL, and for N-terminal pro-BNP (NT-proBNP) it is 125 pg/mL; in the acute setting, higher values should be used [e.g., BNP, 100 pg/mL; NT-proBNP, 300 pg/mL; and mid-regional pro A-type natriuretic peptide (MR-proANP), 120 pmol/L]. Diagnostic values apply similarly to HFrEF and HFpEF. On average, values are typically lower for HFpEF than for HFrEF.

There are numerous cardiovascular and non-cardiovascular causes of elevated NPs that may weaken their diagnostic utility in HF. Among them, AF, age and renal failure are the most important factors impeding the interpretation of NP measurements. On the other hand, NP levels may be disproportionally low in obese patients.

BNP

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has elevated levels of BNP. In some embodiments, the method relates to patients having a BNP level of at least 35 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 40 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 50 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 60 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 70 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 80 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 90 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 100 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 150 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 200 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 300 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 400 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 500 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 1000 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 5000 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 10,000 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 15,000 pg/mL. In some embodiments, the method relates to patients having a BNP level of at least 20,000 pg/mL.

In some embodiments, the disclosure relates to methods of adjusting one or more natriuretic peptides in the heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to reducing the patient's BNP by at least 5 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 10 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 50 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 100 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 200 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 500 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 1000 pg/mL. In some embodiments, the method relates to reducing the patient's BNP by at least 5000 pg/mL.

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

NT-proBNP

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has a NT-proBNP level of at least 100 pg/mL (e.g., 100, 125, 150, 200, 300, 400, 500, 1000, 3000, 5000, 10,000, 15,000, 20,000, 25,000, or 30,000 pg/mL). In some embodiments, the method relates to patient's having a NT-proBNP level of at least 100 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 125 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 150 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 200 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 300 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 400 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 500 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 1000 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 5000 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 10,000 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 15,000 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 20,000 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 25,000 pg/mL. In some embodiments, the method relates to patients having a NT-proBNP level of at least 30,000 pg/mL.

In some embodiments, the disclosure relates to methods of adjusting one or more natriuretic peptides in the heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 10 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 25 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 50 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 100 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 200 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 500 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 1000 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 5000 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 10,000 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 15,000 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 20,000 pg/mL. In some embodiments, the method relates to reducing the patient's NT-proBNP by at least 25,000 pg/mL.

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

Troponin Levels

Troponin, or the troponin complex, is a complex of three regulatory proteins (troponin C, troponin I, and troponin T) that is integral to muscle contraction in skeletal muscle and cardiac muscle, but not smooth muscle. Blood troponin levels may be used as a diagnostic marker for stroke, although the sensitivity of this measurement is low. Measurements of cardiac-specific troponins I and T are extensively used as diagnostic and prognostic indicators in the management of myocardial infarction and acute coronary syndrome.

Certain subtypes of troponin (cardiac I and T) are sensitive and specific indicators of damage to the heart muscle (myocardium). They are measured in the blood to differentiate between unstable angina and myocardial infarction (heart attack) in people with chest pain or acute coronary syndrome. A person who recently had a myocardial infarction would have an area of damaged heart muscle and elevated cardiac troponin levels in the blood. This can also occur in people with coronary vasospasm, a type of myocardial infarction involving severe constriction of the cardiac blood vessels. After a myocardial infarction troponins may remain high for up to 2 weeks.

Cardiac troponins are a marker of heart muscle damage. Diagnostic criteria for raised troponin indicating myocardial infarction is currently set by the WHO at a threshold of 2 μg or higher. Critical levels of other cardiac biomarkers are also relevant, such as creatine kinase. Other conditions that directly or indirectly lead to heart muscle damage and death can also increase troponin levels, such as kidney failure. Severe tachycardia (for example due to supraventricular tachycardia) in an individual with normal coronary arteries can also lead to increased troponins for example, it is presumed due to increased oxygen demand and inadequate supply to the heart muscle.

Troponins are increased in patients with heart failure, where they also predict mortality and ventricular rhythm abnormalities. They can rise in inflammatory conditions such as myocarditis and pericarditis with heart muscle involvement (which is then termed myopericarditis). Troponins can also indicate several forms of cardiomyopathy, such as dilated cardiomyopathy, hypertrophic cardiomyopathy or (left) ventricular hypertrophy, peripartum cardiomyopathy, Takotsubo cardiomyopathy, or infiltrative disorders such as cardiac amyloidosis.

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has elevated levels of troponin. In some embodiments, the disclosure relates to methods of adjusting one or more parameters in the heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to decreasing the patient's troponin levels by 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 method relates to decreasing the patient's troponin levels by at least 1%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 5%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 10%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 15%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 20%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 25%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 30%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 35%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 40%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 45%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 50%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 55%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 60%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 65%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 70%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 75%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 80%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 85%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 90%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 95%. In some embodiments, the method relates to decreasing the patient's troponin levels by at least 100%.

Right Ventricular Hypertrophy

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has right ventricular hypertrophy. In some embodiments, the disclosure relates to methods of adjusting one or more parameters in the heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by 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 method relates to decreasing the patient's right ventricular hypertrophy by at least 1%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 5%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 10%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 15%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 20%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 25%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 30%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 35%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 40%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 45%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 50%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 55%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 60%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 65%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 70%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 75%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 80%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 85%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 90%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 95%. In some embodiments, the method relates to decreasing the patient's right ventricular hypertrophy by at least 100%.

Left Ventricular Hypertrophy

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has left ventricular hypertrophy. In some embodiments, the disclosure relates to methods of adjusting one or more parameters in the heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by 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 method relates to decreasing the patient's left ventricular hypertrophy by at least 1%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 5%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 10%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 15%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 20%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 25%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 30%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 35%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 40%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 45%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 50%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 55%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 60%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 65%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 70%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 75%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 80%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 85%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 90%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 95%. In some embodiments, the method relates to decreasing the patient's left ventricular hypertrophy by at least 100%.

Smooth Muscle Hypertrophy

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has left ventricular hypertrophy. In some embodiments, the disclosure relates to methods of adjusting one or more parameters in the heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by 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 method relates to decreasing the patient's smooth muscle hypertrophy by at least 1%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 5%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 10%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 15%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 20%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 25%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 30%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 35%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 40%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 45%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 50%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 55%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 60%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 65%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 70%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 75%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 80%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 85%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 90%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 95%. In some embodiments, the method relates to decreasing the patient's smooth muscle hypertrophy by at least 100%.

Rate of Hospitalization

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method reduces the patient's hospitalization rate (e.g., by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%4, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 1%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 2%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 3%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 4%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 5%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 10%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 15%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 20%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 25%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 30%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 35%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 40%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 45%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 50%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 55%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 60%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 65%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 70%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 75%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 80%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 85%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 90%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 95%. In some embodiments, the method relates to reducing the patient's hospitalization rate by at least 100%.

Rate of Worsening of Heart Failure

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method reduces the patient's rate of worsening of heart failure (e.g., by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 1%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 2%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 3%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 4%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 5%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 10%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 15%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 20%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 25%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 30%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 35%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 40%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 45%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 50%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 55%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 60%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 65%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 70%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 75%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 80%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 85%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 90%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 95%. In some embodiments, the method relates to reducing the patient's rate of worsening of heart failure by at least 100%.

Diastolic Function

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method increases the patient's LV diastolic function (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%). In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 5%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 10%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 15%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 20%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 25%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 30%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 35%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 40%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 45%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 50%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 55%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 60%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 65%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 70%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 75%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 80%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 85%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 90%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 95%. In some embodiments, the method relates to increasing the patient's LV diastolic function by at least 100%.

Ejection Fraction

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient has an ejection fraction of less than 45% (e.g., 10, 15, 20, 25, 30, 35, 40, or 45%). In some embodiments, the method relates to patient's having an ejection fraction of less than 10%. In some embodiments, the method relates to patient's having an ejection fraction of less than 15%. In some embodiments, the method relates to patient's having an ejection fraction of less than 20%. In some embodiments, the method relates to patient's having an ejection fraction of less than 25%. In some embodiments, the method relates to patient's having an ejection fraction of less than 30%. In some embodiments, the method relates to patient's having an ejection fraction of less than 35%. In some embodiments, the method relates to patient's having an ejection fraction of less than 40%. In some embodiments, the method relates to patient's having an ejection fraction of less than 45%. In some embodiments, the method relates to patient's having an ejection fraction of less than 50%. In some embodiments, the method relates to patient's having an ejection fraction of less than 55%. In some embodiments, the ejection fraction is the right ventricular ejection fraction. In some embodiments, the ejection fraction is the left ventricular ejection fraction. In some embodiments, the ejection fraction is measured using an echocardiogram. In some embodiments, the patient has a preserved left ventricular ejection fraction.

In some embodiments, the disclosure relates to methods increasing ejection fraction in a heart failure patient toward a more normal level (e.g., normal as compared to healthy people of similar age and sex), comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the method relates to increasing the patient's ejection fraction by least 1%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 5%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 10%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 15%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 20%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 25%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 30%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 35%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 40%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 45%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 50%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 55%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 60%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 65%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 70%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 75%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 80%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 85%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 90%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 95%. In some embodiments, the method relates to increasing the patient's ejection fraction by at least 100%.

Cardiac Output

In general, normal cardiac output at rest is about 2.5-4.2 L/min/m2, and cardiac output can decline by almost 40% without deviating from the normal limits. A low cardiac index of less than about 2.5 L/min/m2 usually indicates a disturbance in cardiovascular performance. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the method increases 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%). In some embodiments, the method relates to increasing the patient's cardiac output by at least 5%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 10%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 15%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 20%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 25%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 30%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 35%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 40%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 45%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 50%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 55%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 60%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 65%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 70%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 75%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 80%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 85%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 90%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 95%. In some embodiments, the method relates to increasing the patient's cardiac output by at least 100%. In some embodiments, the method relates to increasing the patient's cardiac output to at least 4.2 L/min/m2. In some embodiments, the cardiac output is measured at rest. In some embodiments, the cardiac output is measured using a right heart catheter.

Exercise Capacity (6MWD AND BDI)

In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). Any suitable measure of exercise capacity can be used. For example, exercise capacity in a 6-minute walk test (6MWT), which measures how far the patient can walk in 6 minutes, i.e., the 6-minute walk distance (6MWD), is frequently used to assess heart failure severity and disease progression. The Borg dyspnea index (BDI) is a numerical scale for assessing perceived dyspnea (breathing discomfort). It measures the degree of breathlessness, for example, after completion of the 6MWT, where a BDI of 0 indicates no breathlessness and 10 indicates maximum breathlessness. In some embodiments, the method relates to increasing 6MWD by at least 10 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 30 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 40 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 60 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 70 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 80 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 90 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relates to increasing 6MWD by at least 100 meters in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the 6MWD is tested after the patient has received 4 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 8 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 12 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 16 weeks of treatment utilizing de disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 20 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 22 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 24 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 26 weeks of treatment utilizing disclosed herein. In some embodiments, the 6MWD is tested after the patient has received 28 weeks of treatment utilizing disclosed herein. In some embodiments, the method relate to lowering BDI by at least 0.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 1 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 1.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 2 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 2.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 3 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 3.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 4 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 4.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 5.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 6 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 6.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 7 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 7.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 8 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 8.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 9 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 9.5 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by at least 3 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, the method relate to lowering BDI by 10 index points in the patient having heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies).

Cardiac Imaging

Echocardiogram

The term “echocardiography” as used herein refers to two-dimensional/three-dimensional echocardiography, pulsed and continuous wave Doppler, color flow Doppler, tissue Doppler imaging (TDI) contrast echocardiography, deformation imaging (strain and strain rate), and transthoracic echocardiography (TTE). TTE is typically the method of choice for assessment of myocardial systolic and diastolic function of both left and right ventricles. In some embodiments, a patient is assessed for heart failure using echocardiography. In some embodiments, a patient is assessed for heart failure using two-dimensional echocardiography. In some embodiments, a patient is assessed for heart failure using three-dimensional echocardiography. In some embodiments, a patient is assessed for heart failure using pulsed and continuous wave Doppler echocardiography. In some embodiments, a patient is assessed for heart failure using echocardiography. In some embodiments, a patient is assessed for heart failure using, color flow Doppler echocardiography. In some embodiments, a patient is assessed for heart failure using tissue Doppler imaging (TDI) contrast echocardiography. In some embodiments, a patient is assessed for heart failure using deformation imaging (strain and strain rate) echocardiography. In some embodiments, a patient is assessed for heart failure using transthoracic echocardiography (TTE).

An abnormal electrocardiogram (ECG) increases the likelihood of a diagnosis of HF, but has low specificity. Some abnormalities on an ECG provide information on etiology (e.g., myocardial infarction), and findings on an ECG might provide indications for therapy (e.g., anticoagulation for AF, pacing for bradycardia, etc.). HF is unlikely in patients presenting with a completely normal ECG (sensitivity 89%) Therefore, routine use of an ECG is mainly recommended to rule out HF. Echocardiography is a useful and widely available test in patients with suspected HF to establish a diagnosis. It provides information on 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 equation FS=100%×[(EDD−ESD)/EDD]), LV end diastolic volume (LVEDV), LV end systolic volume (LVESV), ejection fraction (calculated using the equation 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). This information is crucial in establishing a diagnosis and in determining appropriate treatment. In some embodiments, a patient's LV wall thickness (LVWT) is measured using echocardiography. In some embodiments, a patient's LV mass (LVM) is measured using echocardiography. In some embodiments, a patient's LV end diastolic diameter (LVEDD) is measured using echocardiography. In some embodiments, a patient's LV end systolic diameter (LVESD) is measured using echocardiography. In some embodiments, a patient's fractional shortening (FS) is measured using echocardiography. In some embodiments, a patient's LV end diastolic volume (LVEDV) is measured using echocardiography. In some embodiments, a patient's LV end systolic volume (LVESV) is measured using echocardiography. In some embodiments, a patient's ejection fraction is measured using echocardiography. In some embodiments, a patient's hypertrophy index is measured using echocardiography. In some embodiments, a patient's relative wall thickness is measured using echocardiography. There are numerous clinical presentation factors, echocardiography features, and other features that could be indicative of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies). In some embodiments, an echocardiogram performed on a patient shows structural left heart abnormalities. In some embodiments, the structural left heart abnormality is a disease of the left heart valves. In some embodiments, the structural left heart abnormality is left atrium enlargement (e.g., >4.2 cm). In some embodiments, an electrocardiogram performed on a patient shows left ventricular hypertrophy (LVH) and/or left atrial hypertrophy/dilation (LAH). In some embodiments, an electrocardiogram performed on a patient shows atrial flutter/atrial fibrillation (AF/Afib). In some embodiments, an electrocardiogram performed on a patient shows left bundle branch block (LBBB). In some embodiments, an electrocardiogram performed on a patient shows the presence of Q waves. See, e.g., Galie N., et al Euro Heart J. (2016) 37, 67-119.

In a patient that has symptoms of left heart failure, an echocardiogram may be performed to evaluate various parameters. For instance, in some embodiments, an echocardiogram using Doppler performed on a patient may show indices of increased filling pressures and/or diastolic dysfunction (e.g., increased E/E′ or >Type 2-3 mitral flow abnormality). In some embodiments, imaging (e.g. echocardiogram, CT scan, chest X-ray, or MRI) performed on a patient shows Kerley B lines. In some embodiments, imaging (e.g. echocardiogram, CT scan, chest X-ray, or MRI) performed on a patient shows pleural effusion. In some embodiments, imaging (e.g. echocardiogram, CT scan, chest X-ray, or MRI) performed on a patient shows pulmonary edema. In some embodiments, imaging (e.g., echocardiogram, CT scan, chest X-ray, or MRI) performed on a patient shows left atrium enlargement. Id.

Key structural alterations in HFpEF/HFmrEF heart failure comprise a left atrial volume index (LAVI)>34 mL/m2 and/or a left ventricular mass index (LVMI)≥115 g/m2 for males and ≥95 g/m2 for females.

Key functional alterations of HFpEF/HFmEF heart failure comprise an E/e′≥13 and a mean e′ septal and lateral wall<9 cm/s. Other (indirect) echocardiographically derived measurements are longitudinal strain or tricuspid regurgitation velocity (TRV).

Echocardiography examination may also include assessment of right ventricle (RV) structure and function, including, but not limited to, RV and right atrial (RA) dimensions, and an estimation of RV systolic function and/or pulmonary arterial pressure. Among parameters reflecting RV systolic function, the following measures are of particular importance: tricuspid annular plane systolic excursion (TAPSE; abnormal TAPSE<17 mm indicates RV systolic dysfunction) and tissue Doppler-derived tricuspid lateral annular systolic velocity (s′) (s′ velocity<9.5 cm/s indicates RV systolic dysfunction). Systolic pulmonary artery pressure is derived from an optimal recording of maximal tricuspid regurgitant jet and the tricuspid systolic gradient, together with an estimate of RA pressure on the basis of inferior vena cava (IVC) size and its breathing-related collapse. Exercise or pharmacological stress echocardiography may be used for the assessment of inducible ischemia and/or myocardium viability and in some clinical scenarios of patients with valve disease (e.g. dynamic mitral regurgitation, low-flow-low-gradient aortic stenosis). There are also suggestions that stress echocardiography may allow the detection of diastolic dysfunction related to exercise exposure in patients with exertional dyspnea, preserved LVEF, and inconclusive diastolic parameters at rest.

Transthoracic echocardiography (TTE) is recommended for the assessment of myocardial structure and function in patients with suspected HF in order to establish a diagnosis of either HFrEF, HFmrEF or HFpEF. Furthermore, TTE is recommended to assess LVEF in order to identify patients with HF who would be suitable for evidence-based pharmacological and device (ICD, CRT) treatment recommended for HFrEF; for the assessment of valve disease, right ventricular function and pulmonary arterial pressure in patients with an already established diagnosis of either HFrEF, HFmrEF or HFpEF in order to identify those suitable for correction of valve disease; and/or for the assessment of myocardial structure and function in patients to be exposed to treatment which potentially can damage myocardium (e.g. chemotherapy). Other techniques (including systolic tissue Doppler velocities and deformation indices, i.e. strain and strain rate), should be considered in a TTE protocol in patients at risk of developing HF in order to identify myocardial dysfunction at the preclinical stage.

Cardiac Magnetic Resonance (CMR)

CMR is acknowledged as a gold standard for the measurements of volumes, mass and EF of both the left and right ventricles. It is the best alternative cardiac imaging modality for patients with nondiagnostic echocardiographic studies (particularly for imaging of the right heart) and is the method of choice in patients with complex congenital heart diseases. Cardiac magnetic resonance (CMR) measures both cardiac anatomical and functional quantification, with unique capabilities of non-invasive tissue characterization, complementing well with echocardiography. CMR imaging covering the LV in short axis from apex to base is used for measuring left ventricular (LV) volumes, ejection fraction (EF) and regional function. The 3D dataset is not affected by geometric assumptions and therefore less prone to error compared with two-dimensional (2D) echocardiography, particularly in remodeled ventricles. Novel CMR tissue characterization techniques are called CMR relaxometry (T1 and T2 mapping and extracellular volume fraction (ECV)) which allow a more detailed and quantitative approach to tissue characterization and 4D-Flow which provides quantitative information on intracavitary flows. Current applications appear particularly useful for diastolic dysfunction detection although they deserve a specific comparison with traditional Doppler and Tissue Doppler (e.g., echocardiography) analysis in order to confirm the applicability in clinical practice. Non-invasive stress imaging (CMR, stress echocardiography, SPECT, PET) may be considered for the assessment of myocardial ischemia and viability in patients with HF and CAD (considered suitable for coronary revascularization) before the decision on revascularization. In some embodiments, a patient is assessed for heart failure using CMR. In some embodiments, a patient is assessed for heart failure using CMR relaxometry (T1 and T2 mapping and extracellular volume fraction (ECV)). In some embodiments, a patient is assessed for heart failure using CMR and 4D-Flow.

CMR can provide information on LV structure and systolic function, 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 equation FS=100%×[(EDD−ESD)/EDD]), LV end diastolic volume (LVEDV), LV end systolic volume (LVESV), ejection fraction (calculated using the equation 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). This information is crucial in establishing a diagnosis and in determining appropriate treatment. In some embodiments, a patient's LV wall thickness (LVWT) is measured using CMR. In some embodiments, a patient's LV mass (LVM) is measured using CMR. In some embodiments, a patient's LV end diastolic diameter (LVEDD) is measured using CMR. In some embodiments, a patient's LV end systolic diameter (LVESD) is measured using CMR. In some embodiments, a patient's fractional shortening (FS) is measured using CMR. In some embodiments, a patient's LV end diastolic volume (LVEDV) is measured using CMR. In some embodiments, a patient's LV end systolic volume (LVESV) is measured using CMR. In some embodiments, a patient's ejection fraction is measured using CMR. In some embodiments, a patient's hypertrophy index is measured using CMR. In some embodiments, a patient's relative wall thickness is measured using CMR.

CMR is a preferred imaging method to assess myocardial fibrosis using late gadolinium enhancement (LGE) along with T1 mapping and can be useful for establishing HF etiology. For example, CMR with LGE allows differentiation between ischemic and non-ischemic origins of HF and myocardial fibrosis/scars can be visualized. In addition, CMR allows the characterization of myocardial tissue of myocarditis, amyloidosis, sarcoidosis, Chagas disease, Fabry disease non-compaction cardiomyopathy and haemochromatosis. CMR may also be used for the assessment of myocardial ischemia and viability in patients with HF and coronary artery disease (CAD) (considered suitable for coronary revascularization). In some embodiments, a patient is assessed for heart failure using CMR with late gadolinium enhancement (LGE) and/or T1 mapping. In some embodiments, fibrosis and/or scars in a patient's heart is measured using CMR.

Clinical limitations of CMR include local expertise, lower availability and higher costs compared with echocardiography, uncertainty about safety in patients with metallic implants (including cardiac devices) and less reliable measurements in patients with tachyarrhythmias. Claustrophobia is an important limitation for CMR. Linear gadolinium-based contrast agents are contraindicated in individuals with a glomerular filtration rate (GFR)<30 mL/min/1.73 m², because they may trigger nephrogenic systemic fibrosis (this may be less of a concern with newer cyclic gadolinium-based contrast agents).

CMR is recommended for the assessment of myocardial structure and function (including right heart) in patients with poor acoustic window and patients with complex congenital heart diseases (taking account of cautions/contra-indications to CMR). CMR with LGE should be considered in patients with dilated cardiomyopathy in order to distinguish between ischemic and nonischemic myocardial damage in case of equivocal clinical and other imaging data (taking account of cautions/contra-indications to CMR). CMR is recommended for the characterization of myocardial tissue in case of suspected myocarditis, amyloidosis, sarcoidosis, Chagas disease, Fabry disease non-compaction cardiomyopathy, and haemochromatosis (taking account of cautions/contraindications to CMR).

Multigated Acquisition (MUGA)

Radionuclide angiography is an area of nuclear medicine which specializes in imaging to show the functionality of the right and left ventricles of the heart, thus allowing informed diagnostic intervention in heart failure. It involves use of a radiopharmaceutical injected into a patient, and a gamma camera for acquisition. A MUGA scan (multigated acquisition) involves an acquisition triggered (gated) at different points of the cardiac cycle. MUGA scanning is also sometimes referred to as equilibrium radionuclide angiocardiography, radionuclide ventriculography (RNVG), or gated blood pool imaging, as well as SYMA scanning (synchronized multigated acquisition scanning). In some embodiments, a patient is assessed for heart failure using MUGA. In some embodiments, a patient is assessed for heart failure using equilibrium radionuclide angiocardiography. In some embodiments, a patient is assessed for heart failure using radionuclide ventriculography (RNVG). In some embodiments, a patient is assessed for heart failure using gated blood pool imaging. In some embodiments, a patient is assessed for heart failure using SYMA scanning (synchronized multigated acquisition scanning).

MUGA uniquely provides a cine type of image (e.g., short movies that are able to show heart motion throughout the cardiac cycle) of the beating heart, and allows the interpreter to determine the efficiency of the individual heart valves and chambers. MUGA/Cine scanning represents a robust adjunct to an echocardiogram. Mathematics regarding acquisition of cardiac output (Q) is well served by both of these methods as well as other inexpensive models supporting ejection fraction as a product of the heart/myocardium in systole. One main advantage of a MUGA scan over an echocardiogram or an angiogram is its accuracy. An echocardiogram measures the shortening fraction of the ventricle and is limited by the user's ability. Furthermore, an angiogram is invasive and, often, more expensive. A MUGA scan provides a more accurate representation of cardiac ejection fraction.

Chest X-Ray

A chest X-ray is of limited use in the diagnostic work-up of patients with suspected HF. It is most useful in identifying an alternative, pulmonary explanation for a patient's symptoms and signs, (e.g., pulmonary malignancy and/or interstitial pulmonary disease), although computed tomography (CT) of the chest is currently the standard of care for these types of pulmonary diseases. For diagnosis of asthma or chronic obstructive pulmonary disease (COPD), pulmonary function testing with spirometry is needed. A chest X-ray may, however, show pulmonary venous congestion or edema in a patient with HF, and is more helpful in the acute setting than in the non-acute setting. In some embodiments, a patient is assessed for heart failure using chest X-ray.

Single-Photon Emission Computed Tomography (SPECT) and Radionucleotide Ventriculography

Single-photon emission CT (SPECT) may be useful in assessing ischemia and myocardial viability. Gated SPECT can also yield information on ventricular volumes and function, but exposes the patient to ionizing radiation. 3,3-diphosphono-1,2-propanodicarboxylic acid (DPD) scintigraphy may be useful for the detection of transthyretin cardiac amyloidosis. In some embodiments, a patient is assessed for heart failure using SPECT.

Positron Emission Tomography (PET)

Positron emission tomography (PET) (alone or with CT) may be used to assess ischemia and viability, but flow tracers (N-13 ammonia or O-15 water) require an on-site cyclotron. Rubidium is an alternative tracer for ischemia testing with PET, which can be produced locally at relatively low cost. Limited availability, radiation exposure and cost are the main limitations. In some embodiments, a patient is assessed for heart failure using PET.

Coronary Angiography

Coronary angiography is recommended in patients with HF who suffer from angina pectoris recalcitrant to medical therapy, provided the patient is otherwise suitable for coronary revascularization. Coronary angiography is also recommended in patients with a history of symptomatic ventricular arrhythmia or aborted cardiac arrest. Coronary angiography should be considered in patients with HF and intermediate to high pre-test probability of coronary artery disease (CAD) and the presence of ischemia in non-invasive stress tests in order to establish the ischemic etiology and CAD severity. In some embodiments, a patient is assessed for heart failure using coronary angiography.

Invasive coronary angiography is recommended in patients with HF and angina pectoris recalcitrant to pharmacological therapy or symptomatic ventricular arrhythmias or aborted cardiac arrest (who are considered suitable for potential coronary revascularization) in order to establish the diagnosis of CAD and its severity. Invasive coronary angiography should be considered in patients with HF and intermediate to high pre-test probability of CAD and the presence of ischemia in non-invasive stress tests (who are considered suitable for potential coronary revascularization) in order to establish the diagnosis of CAD and its severity.

Cardiac Computer Tomography (CT)

The main use of cardiac CT in patients with HF is as a non-invasive means to visualize the coronary anatomy in patients with HF with low intermediate pre-test probability of coronary artery disease (CAD) or those with equivocal non-invasive stress tests in order to exclude the diagnosis of CAD, in the absence of relative contraindications. However, the test is only required when its results might affect a therapeutic decision. Cardiac CT may be considered in patients with HF and low to intermediate pre-test probability of CAD or those with equivocal non-invasive stress tests in order to rule out coronary artery stenosis. In some embodiments, a patient is assessed for heart failure using cardiac computer tomography

Measuring Hematologic Parameters in a Patient

In certain embodiments, the present disclosure provides methods for managing a patient that has been treated with, or is a candidate to be treated with, one or more one or more ActRII-ALK4 antagonists of the disclosure (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) by measuring one or more hematologic parameters in the patient. The hematologic parameters may be used to evaluate appropriate dosing for a patient who is a candidate to be treated with one or more ActRII-ALK4 antagonists of the present disclosure, to monitor the hematologic parameters during treatment, to evaluate whether to adjust the dosage during treatment with one or more ActRII-ALK4 antagonists of the disclosure, and/or to evaluate an appropriate maintenance dose of one or more ActRII-ALK4 antagonists of the disclosure. I_f one or more of the hematologic parameters are outside the normal level, dosing with one or more ActRII-ALK4 antagonists may be reduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with the methods provided herein include, for example, red blood cell levels, blood pressure, iron stores, and other agents found in bodily fluids that correlate with increased red blood cell levels, using art recognized methods. Such parameters may be determined using a blood sample from a patient. Increases in red blood cell levels, hemoglobin levels, and/or hematocrit levels may cause increases in blood pressure.

In one embodiment, if one or more hematologic parameters are outside the normal range or on the high side of normal in a patient who is a candidate to be treated with one or more ActRII-ALK4 antagonists, then onset of administration of the one or more ActRII-ALK4 antagonists of the disclosure may be delayed until the hematologic parameters have returned to a normal or acceptable level either naturally or via therapeutic intervention. For example, if a candidate patient is hypertensive or pre-hypertensive, then the patient may be treated with a blood pressure lowering agent in order to reduce the patient's blood pressure. Any blood pressure lowering agent appropriate for the individual patient's condition may be used including, for example, diuretics, adrenergic inhibitors (including alpha blockers and beta blockers), vasodilators, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensin II receptor blockers. Blood pressure may alternatively be treated using a diet and exercise regimen. Similarly, if a candidate patient has iron stores that are lower than normal, or on the low side of normal, then the patient may be treated with an appropriate regimen of diet and/or iron supplements until the patient's iron stores have returned to a normal or acceptable level. For patients having higher than normal red blood cell levels and/or hemoglobin levels, then administration of the one or more ActRII-ALK4 antagonists of the disclosure may be delayed until the levels have returned to a normal or acceptable level.

In certain embodiments, if one or more hematologic parameters are outside the normal range or on the high side of normal in a patient who is a candidate to be treated with one or more ActRII-ALK4 antagonists, then the onset of administration may not be delayed. However, the dosage amount or frequency of dosing of the one or more ActRII-ALK4 antagonists of the disclosure may be set at an amount that would reduce the risk of an unacceptable increase in the hematologic parameters arising upon administration of the one or more ActRII-ALK4 antagonists of the disclosure. Alternatively, a therapeutic regimen may be developed for the patient that combines one or more ActRII-ALK4 antagonists with a therapeutic agent that addresses the undesirable level of the hematologic parameter. For example, if the patient has elevated blood pressure, then a therapeutic regimen may be designed involving administration of one or more ActRII-ALK4 antagonists and a blood pressure lowering agent. For a patient having lower than desired iron stores, a therapeutic regimen may be developed involving one or more ActRII-ALK4 antagonists of the disclosure and iron supplementation.

In one embodiment, baseline parameter(s) for one or more hematologic parameters may be established for a patient who is a candidate to be treated with one or more ActRII-ALK4 antagonists of the disclosure and an appropriate dosing regimen established for that patient based on the baseline value(s). Alternatively, established baseline parameters based on a patient's medical history could be used to inform an appropriate ActRII-ALK4 antagonist dosing regimen for a patient. For example, if a healthy patient has an established baseline blood pressure reading that is above the defined normal range it may not be necessary to bring the patient's blood pressure into the range that is considered normal for the general population prior to treatment with the one or more ActRII-ALK4 antagonists of the disclosure. A patient's baseline values for one or more hematologic parameters prior to treatment with one or more ActRII-ALK4 antagonists of the disclosure may also be used as the relevant comparative values for monitoring any changes to the hematologic parameters during treatment with the one or more ActRII-ALK4 antagonists of the disclosure.

In certain embodiments, one or more hematologic parameters are measured in patients who are being treated with one or more ActRII-ALK4 antagonists. The hematologic parameters may be used to monitor the patient during treatment and permit adjustment or termination of the dosing with the one or more ActRII-ALK4 antagonists of the disclosure or additional dosing with another therapeutic agent. For example, if administration of one or more ActRII-ALK4 antagonists results in an increase in blood pressure, red blood cell level, or hemoglobin level, or a reduction in iron stores, then the dose of the one or more ActRII-ALK4 antagonists of the disclosure may be reduced in amount or frequency in order to decrease the effects of the one or more ActRII-ALK4 antagonists of the disclosure on the one or more hematologic parameters. If administration of one or more ActRII-ALK4 antagonists results in a change in one or more hematologic parameters that is adverse to the patient, then the dosing of the one or more ActRII-ALK4 antagonists of the disclosure may be terminated either temporarily, until the hematologic parameter(s) return to an acceptable level, or permanently. Similarly, if one or more hematologic parameters are not brought within an acceptable range after reducing the dose or frequency of administration of the one or more ActRII-ALK4 antagonists of the disclosure, then the dosing may be terminated. As an alternative, or in addition to, reducing or terminating the dosing with the one or more ActRII-ALK4 antagonists of the disclosure, the patient may be dosed with an additional therapeutic agent that addresses the undesirable level in the hematologic parameter(s), such as, for example, a blood pressure lowering agent or an iron supplement. For example, if a patient being treated with one or more ActRII-ALK4 antagonists has elevated blood pressure, then dosing with the one or more ActRII-ALK4 antagonists of the disclosure may continue at the same level and a blood-pressure-lowering agent is added to the treatment regimen, dosing with the one or more antagonist of the disclosure may be reduced (e.g., in amount and/or frequency) and a blood-pressure-lowering agent is added to the treatment regimen, or dosing with the one or more antagonist of the disclosure may be terminated and the patient may be treated with a blood-pressure-lowering agent.

8. Additional Treatments for Heart Failure and Co-Therapies

In certain aspects, the disclosure contemplates the use of an ActRII-ALK4 antagonist, in combination with one or more additional active agents or other supportive therapy for treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) As used herein, “in combination with”, “combinations of”, “combined with”, or “conjoint” administration refers to any form of administration such that additional active agents or supportive therapies (e.g., second, third, fourth, etc.) are still effective in the body (e.g., multiple compounds are simultaneously effective in the patient for some period of time, which may include synergistic effects of those compounds). Effectiveness may not correlate to measurable concentration of the agent in blood, serum, or plasma. For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially, and on different schedules. Thus, a subject who receives such treatment can benefit from a combined effect of different active agents or therapies. One or more ActRII-ALK4 antagonists of the disclosure can be administered concurrently with, prior to, or subsequent to, one or more other additional agents or supportive therapies, such as those disclosed herein. In general, each active agent or therapy will be administered at a dose and/or on a time schedule determined for that particular agent. The particular combination to employ in a regimen will take into account compatibility of the ActRII-ALK4 antagonist of the disclosure with the additional active agent or therapy and/or the desired effect.

Some goals of treatment in patients with HF is to improve their clinical status, functional capacity and quality of life, and/or prevent hospital admission and reduce mortality. Neuro-hormonal antagonists (e.g., ACEIs, MRAs and beta-blockers) have been shown to improve survival in patients with HFrEF and have been recommended for the treatment of patients with HFrEF, unless contraindicated or not tolerated. In certain aspects, the disclosure relates to methods of treating, preventing, or reducing the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist), wherein the patient is also administered one or more of an angiotensin-converting enzyme inhibitor (ACE inhibitor), beta-blocker, angiotensin II receptor blocker (ARB), Mineralcorticoid/aldosterone receptor antagonist (MRA) or implantable cardioverter defibrillator (ICD). In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and an angiotensin-converting enzyme inhibitor (ACEI) to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and a beta-blocker to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and an angiotensin II receptor blocker (ARB) to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and a mineralcorticoid/aldosterone receptor antagonist (MRA) to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and an implantable cardioverter defibrillator (ICD) to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and an angiotensin receptor neprilysin inhibitor (ARNI) to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and a diuretic to a patient in need thereof. In some embodiments, the method relates to administering an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) and one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), and I_f-channel inhibitor (e.g., Ivabradine) to a patient in need thereof.

Optionally, methods disclosed herein for treating, preventing, or reducing the progression rate and/or severity of heart failure, particularly treating, preventing, or reducing the progression rate and/or severity of one or more comorbidities of heart failure, may further comprise administering to the patient one or more supportive therapies or additional active agents for treating heart failure. For example, the patient also may be administered one or more supportive therapies or active agents selected from the group consisting of: ACE inhibitors (e.g., benazepril, captopril, enalapril, lisinopril, perindopril, ramipril (e.g., ramipen), trandolapril, and zofenopril); beta blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, and timolol); ARBs (e.g., losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan, azilsartan, salprisartan, and telmisartan); mineralcorticoid/aldosterone receptor antagonists (MRAs) (e.g., progesterone, eplerenone and spironolactone); glucocorticoids (e.g., beclomethasone, betamethasone, budesonide, cortisone, deflazacort, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, methylprednisone, prednisone, triamcinolone, and finerenone); statins (e.g., atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pravastatin (Pravachol), pitavastatin (Livalo), simvastatin (Zocor), and rosuvastatin (Crestor)); Sodium-glucose co-transporter 2 (SGLT2) inhibitors (e.g., canagliflozin, dapagliflozin (e.g., Farxiga), and empagliflozin); an implantable cardioverter defibrillator (ICD); angiotensin receptor neprilysin inhibitors (ARNI) (e.g., valsartan and sacubitril (a neprilysin inhibitor)); diuretics (e.g., furosemide, bumetanide, torasemide, bendroflumethiazide, hydrochlorothiazide, metolazone, indapamidec, spironolactone/eplerenone, amiloride and triamterene); and other therapies including hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), and I_f-channel inhibitor (e.g., Ivabradine).

Angiotensin-Converting Enzyme (ACE) Inhibitors

An ACE inhibitor is recommended in patients with asymptomatic LV systolic dysfunction and a history of myocardial infarction in order to prevent or delay the onset of HF and prolong life, or in patients with asymptomatic LV systolic dysfunction without a history of myocardial infarction, in order to prevent or delay the onset of HF. ACE inhibitors should be considered in patients with stable CAD even if they do not have LV systolic dysfunction, in order to prevent or delay the onset of HF. ACE inhibitors have been shown to reduce mortality and morbidity in patients with HFrEF, and are recommended unless contraindicated or not tolerated in all symptomatic patients.

In some embodiments, the disclosure relates to a method of treating a patient with heart failure by administering an ACE inhibitor. In some embodiments, an ACE inhibitor is selected from the group consisting of benazepril, captopril, enalapril, lisinopril, perindopril, ramipril (e.g., ramipen), trandolapril, and zofenopril. In some embodiments, a patient is administered benazepril. In some embodiments, a patient is administered captopril. In some embodiments, a patient is administered enalapril. In some embodiments, a patient is administered lisinopril. In some embodiments, a patient is administered perindopril. In some embodiments, a patient is administered ramipril. In some embodiments, a patient is administered trandolapril. In some embodiments, a patient is administered zofenopril. In some embodiments, administration of an ACE inhibitor

In some embodiments, administration of an ACE inhibitor delays the onset of heart failure in a patient. In some embodiments, administration of an ACE inhibitor prevents the onset of heart failure in a patient. In some embodiments, administration of an ACE inhibitor increases length of life in a patient. In some embodiments, administration of an ACE inhibitor decreases length of a hospital stay in a patient. In some embodiments, administration of an ACE inhibitor prevents hospitalization of a patient.

Beta Blockers

A beta-blocker is recommended in patients with asymptomatic LV systolic dysfunction and a history of myocardial infarction, in order to prevent or delay the onset of HF or prolong life. Beta-blockers can reduce mortality and morbidity in symptomatic patients with HFrEF, despite treatment with an ACEI and, in most cases, a diuretic, but have not been tested in congested or decompensated patients. There is consensus that beta-blockers and ACEIs are complementary, and can be started together as soon as the diagnosis of HFrEF is made.

In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering one or more beta blockers. In some embodiments, the one or more beta blockers is selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, and timolol. In some embodiments a patient is administered acebutolol. In some embodiments, a patient is administered atenolol. In some embodiments, a patient is administered betaxolol. In some embodiments, a patient is administered bisoprolol. In some embodiments, a patient is administered carteolol. In some embodiments, a patient is administered carvedilol. In some embodiments, a patient is administered labetalol. In some embodiments, a patient is administered metoprolol. In some embodiments, a patient is administered nadolol. In some embodiments, a patient is administered nebivolol. In some embodiments, a patient is administered penbutolol. In some embodiments, a patient is administered pindolol. In some embodiments, a patient is administered propranolol. In some embodiments, a patient is administered sotalol. In some embodiments, a patient is administered timolol.

In some embodiments, a patient is administered a beta blocker when the patient shows signs of heart failure. In some embodiments, a patient is administered a beta blocker when the patient is intolerant of ACE inhibitors. In some embodiments, a beta blocker delays onset of heart failure in a patient. In some embodiments, a beta blocker prevents onset of heart failure in a patient. In some embodiments, administration of a beta blocker increases length of life in a patient. In some embodiments, administration of a beta blocker decreases length of a hospital stay in a patient. In some embodiments, administration of a beta blocker prevents hospitalization of a patient.

Angiotensin II Receptor Blockers (ARBs)

Angiotensin II receptor blockers (ARBs) are an alternative in patients who may be intolerant of an ACE inhibitor. Candesartan has been shown to reduce cardiovascular mortality. Valsartan has showed an effect on hospitalization for HF (but not on all-cause hospitalizations) in patients with HFrEF receiving background ACEIs.

In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering one or more ARBs. In some embodiments the one or more ARBs is selected from the group consisting of losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan, azilsartan, salprisartan, and telmisartan. In some embodiments a patient is administered losartan. In some embodiments, a patient is administered irbesartan. In some embodiments, a patient is administered olmesartan. In some embodiments, a patient is administered candesartan. In some embodiments, a patient is administered valsartan. In some embodiments, a patient is administered fimasartan. In some embodiments, a patient is administered azilsartan. In some embodiments, a patient is administered salprisartan. In some embodiments, a patient is administered telmisartan.

In some embodiments, a patient is administered an angiotensin antagonist (e.g., angiotensin receptor blocker, ARB), when the patient shows signs of heart failure. In some embodiments, a patient is administered an ARB when the patient is intolerant of ACE inhibitors. In some embodiments, an ARB delays onset of heart failure in a patient. In some embodiments, an ARB prevents onset of heart failure in a patient. In some embodiments, administration of an ARB increases length of life in a patient. In some embodiments, administration of an ARB decreases length of a hospital stay in a patient. In some embodiments, administration of an ARB prevents hospitalization of a patient.

Corticosteroids

Mineralcorticoid/aldosterone receptor antagonists (MRAs) block receptors that bind aldosterone and, with different degrees of affinity, other steroid hormone receptors (e.g. corticosteroids, androgens). Spironolactone or eplerenone are recommended in symptomatic heart failure patients (despite treatment with an ACE inhibitor and/or beta-blocker) with HFrEF and LVEF≤35%, to reduce mortality and HF hospitalization.

In some embodiments, the disclosure relates to a method of treating a patient with heart failure by administering a corticosteroid. In some embodiments, the patient is administered a Mineralcorticoid/aldosterone receptor antagonist (MRA). In some embodiments, the patient is administered a glucocorticoid. In some embodiments, a patient is administered one or more mineralcorticoid/aldosterone receptor antagonists (MRAs) selected from the group consisting of progesterone, eplerenone and spironolactone. In some embodiments a patient is administered eplerenone. In some embodiments, a patient is administered spironolactone.

In some embodiments, a patient is administered an MRA when the patient shows signs of heart failure. In some embodiments, an MRA delays onset of heart failure in a patient. In some embodiments, an MRA prevents onset of heart failure in a patient. In some embodiments, administration of an MRA increases length of life in a patient. In some embodiments, administration of an MRA decreases length of a hospital stay in a patient. In some embodiments, administration of an MRA prevents hospitalization of a patient.

In some embodiments, a patient with heart failure is administered one or more glucocorticoids. In some embodiments, administration of a glucocorticoid is an initial therapy. In some embodiments, a glucocorticoid is selected from the group consisting of beclomethasone, betamethasone, budesonide, cortisone, deflazacort, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, methylprednisone, prednisone, triamcinolone, and finerenone. In some embodiments, a patient with heart failure is administered prednisone. In some embodiments, a patient with heart failure is administered prednisolone. In some embodiments, a patient with heart failure is administered finerenone. In some embodiments, a patient with heart failure is administered deflazacort.

In some embodiments, a patient is administered a glucocorticoid when the patient shows signs of heart failure. In some embodiments, a glucocorticoid delays onset of heart failure in a patient. In some embodiments, a glucocorticoid prevents onset of heart failure in a patient. In some embodiments, administration of a glucocorticoid increases length of life in a patient. In some embodiments, administration of a glucocorticoid decreases length of a hospital stay in a patient. In some embodiments, administration of a glucocorticoid prevents hospitalization of a patient.

Statins

Treatment with statins is recommended in patients with or at high-risk of CAD whether or not they have LV systolic dysfunction, in order to prevent or delay the onset of HF and prolong life.

In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering one or more statins. In some embodiments, the one or more statins is selected from the group consisting of: atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pravastatin (Pravachol), pitavastatin (Livalo), simvastatin (Zocor), and rosuvastatin (Crestor). In some embodiments a patient is administered atorvastatin. In some embodiments a patient is administered fluvastatin. In some embodiments a patient is administered lovastatin. In some embodiments a patient is administered pravastatin. In some embodiments a patient is administered pitavastatin. In some embodiments a patient is administered simvastatin. In some embodiments a patient is administered rosuvastatin.

In some embodiments, a patient is administered a statin when the patient shows signs of heart failure. In some embodiments, a patient is administered a statin when the patient is at high risk of coronary artery disease (CAD). In some embodiments, a patient is administered a statin when the patient has coronary artery disease (CAD). In some embodiments, a statin delays onset of heart failure in a patient. In some embodiments, a statin prevents onset of heart failure in a patient. In some embodiments, administration of a statin increases length of life in a patient. In some embodiments, administration of a statin decreases length of a hospital stay in a patient. In some embodiments, administration of a statin prevents hospitalization of a patient.

Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitors

Sodium-glucose co-transporter 2 (SGLT2) inhibitors are typically administered along with diet and exercise to lower blood sugar in adults with type 2 diabetes. SGLT2 inhibitors lower blood sugar by causing the kidneys to remove sugar from the body through the urine. Treatment with SGCT2 inhibitors is recommended in patients with heart failure with reduced ejection fraction (HFrEF) to reduce the risk of cardiovascular death and hospitalization for heart failure.

In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering one or more SGLT2 inhibitor. In some embodiments, an SGLT2 inhibitor is a gliflozin. In some embodiments, a patient is administered one or more SGLT2 inhibitors selected from the group consisting of: canagliflozin, dapagliflozin (e.g., Farxiga), and empagliflozin. In some embodiments a patient is administered canagliflozin. In some embodiments a patient is administered dapagliflozin (e.g., Farxiga). In some embodiments a patient is administered empagliflozin.

In some embodiments, a patient is administered an SGLT2 inhibitor when the patient shows signs of heart failure. In some embodiments, a patient is administered an SGLT2 inhibitor when the patient does not have type 2 diabetes. In some embodiments, a patient is administered an SGLT2 inhibitor when the patient has type 2 diabetes. In some embodiments, an SGLT2 inhibitor delays onset of heart failure in a patient. In some embodiments, an SGLT2 inhibitor prevents onset of heart failure in a patient. In some embodiments, administration of an SGLT2 inhibitor increases length of life in a patient. In some embodiments, administration of an SGLT2 inhibitor decreases length of a hospital stay in a patient. In some embodiments, administration of an SGLT2 inhibitor prevents hospitalization of a patient. In some embodiments, an SGLT2 inhibitor reduces the risk of death of a patient.

Implantable Cardioverter Defibrillator (ICD)

Implantable cardioverter defibrillator (ICD) is recommended in patients with one or more of a) asymptomatic LV systolic dysfunction ((e.g., LVEF≤30%) of ischemic origin, who are at least 40 days after acute myocardial infarction, and b) asymptomatic non-ischemic dilated cardiomyopathy (e.g., LVEF≤30%), who receive osteopathic manipulative treatment (OMT), in order to prevent sudden death and prolong life. In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering an implantable cardioverter defibrillator (ICD).

In some embodiments, a patient is administered an ICD when the patient shows signs of heart failure. In some embodiments, a patient with asymptomatic LV systolic dysfunction (e.g., LVEF≤30%) of ischemic origin, who is at least 40 days after acute myocardial infarction, is administered an ICD. In some embodiments, a patient with asymptomatic LV systolic dysfunction (e.g., LVEF≤30%) of ischemic origin is administered an ICD. In some embodiments, a patient who is at least 40 days after acute myocardial infarction is administered an ICD. In some embodiments, a patient with asymptomatic non-ischemic dilated cardiomyopathy (e.g., LVEF≤30%), who receives optimal medical therapy (OMT) is administered an ICD. In some embodiments, a patient with asymptomatic non-ischemic dilated cardiomyopathy (e.g., LVEF≤30%) is administered an ICD. In some embodiments, a patient who receives optimal medical therapy is administered an ICD. In some embodiments, an ICD delays onset of heart failure in a patient. In some embodiments, an ICD prevents onset of heart failure in a patient. In some embodiments, administration of an ICD increases length of life in a patient. In some embodiments, administration of an ICD decreases length of a hospital stay in a patient. In some embodiments, administration of an ICD prevents hospitalization of a patient.

Angiotensin Receptor Neprilysin Inhibitor

A relatively new therapeutic class of agents acting on the renin-angiotensin-aldosterone system (RAAS) and the neutral endopeptidase system has been developed called angiotensin receptor neprilysin inhibitor (ARNI). The first in class is LCZ696, which is a molecule that combines the moieties of valsartan and sacubitril (a neprilysin inhibitor) in a single substance. By inhibiting neprilysin, the degradation of natriuretic peptides (NPs), bradykinin and other peptides is slowed.

High circulating A-type natriuretic peptide (ANP) and BNP exert physiologic effects through binding to NP receptors and the augmented generation of cGMP, thereby enhancing diuresis, natriuresis and myocardial relaxation and anti-remodeling. ANP and BNP also inhibit renin and aldosterone secretion. Selective AT1-receptor blockade reduces vasoconstriction, sodium and water retention and myocardial hypertrophy.

In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering an angiotensin-receptor neprilysin inhibitor. In some embodiments, a patient is administered sacubitril/valsaratan (e.g. LCZ696, Entresto). In some embodiments, a patient with ambulatory, symptomatic HFrEF with LVEF≤35% is administered sacubitril/valsaratan. In some embodiments, a patient with elevated plasma NP levels (BNP≥150 pg/mL and/or NT-proBNP≥600 pg/mL (or, if they had been hospitalized for HF within the previous 12 months, BNP≥100 pg/mL and/or NT-proBNP≥400 pg/mL) is administered sacubitril/valsaratan. In some embodiments, a patient with ambulatory, symptomatic HFrEF with LVEF≤35% is administered sacubitril/valsaratan. In some embodiments, a patient with an estimated GFR (eGFR)≥30 mL/min/1.73 m2 of body surface area is administered sacubitril/valsaratan.

In some embodiments, a patient is administered sacubitril/valsartan when the patient shows signs of heart failure. In some embodiments, a patient is administered sacubitril/valsartan when the patient is intolerant of ACE inhibitors. In some embodiments, a patient is administered sacubitril/valsartan when the patient is intolerant of beta blockers. In some embodiments, a patient is administered sacubitril/valsartan when the patient is intolerant of MRAs. In some embodiments, a patient is administered sacubitril/valsartan when the patient has HFrEF and remains symptomatic despite treatment with one or more of an ACE inhibitor, a beta-blocker and an MRA. In some embodiments, sacubitril/valsartan delays onset of heart failure in a patient. In some embodiments, sacubitril/valsartan prevents onset of heart failure in a patient. In some embodiments, administration of sacubitril/valsartan increases length of life in a patient. In some embodiments, administration of sacubitril/valsartan decreases length of a hospital stay in a patient. In some embodiments, administration of sacubitril/valsartan prevents hospitalization of a patient.

In some embodiments, a patient is administered an ARNI when the patient shows signs of heart failure. In some embodiments, a patient is administered an ARNI when the patient is intolerant of ACE inhibitors. In some embodiments, a patient is administered an ARNI when the patient is intolerant of beta blockers. In some embodiments, a patient is administered an ARNI when the patient is intolerant of MRAs. In some embodiments, a patient is administered an ARNI when the patient has HFrEF and remains symptomatic despite treatment with one or more of an ACE inhibitor, a beta-blocker and an MRA. In some embodiments, an ARNI delays onset of heart failure in a patient. In some embodiments, an ARNI prevents onset of heart failure in a patient. In some embodiments, administration of an ARNI increases length of life in a patient. In some embodiments, administration of an ARNI decreases length of a hospital stay in a patient. In some embodiments, administration of an ARNI prevents hospitalization of a patient.

Diuretics

Diuretics are recommended to reduce signs and symptoms of congestion in patients with HFrEF. In patients with chronic HF, loop and thiazide diuretics may reduce the risk of death and worsening HF, and also possibly improve exercise capacity. Typically, loop diuretics produce a more intense and shorter diuresis than thiazides, although they act synergistically, and the combination may be used to treat resistant edema.

In some embodiments, the disclosure relates to a method of treating a patient having heart failure by administering one or more diuretics. In some embodiments, a patient is administered one or more diuretics selected from the group consisting of furosemide, bumetanide, torasemide, bendroflumethiazide, hydrochlorothiazide, metolazone, indapamidec, spironolactone/eplerenone, amiloride and triamterene.

In some embodiments, a patient is administered one or more loop diuretics selected from the group consisting of furosemide, bumetanide and torasemide. In some embodiments a patient is administered furosemide. In some embodiments a patient is administered bumetanide. In some embodiments a patient is administered torasemide.

In some embodiments, a patient is administered one or more thiazide diuretics selected from the group consisting of bendroflumethiazide, hydrochlorothiazide, metolazone, and indapamidec. In some embodiments a patient is administered Bendroflumethiazide. In some embodiments a patient is administered hydrochlorothiazide. In some embodiments a patient is administered metolazone. In some embodiments a patient is administered indapamidec.

In some embodiments, a patient is administered one or more potassium-sparing diuretics selected from the group consisting of spironolactone/eplerenone, amiloride and triamterene. In some embodiments a patient is administered spironolactone/eplerenone. In some embodiments a patient is administered amiloride. In some embodiments a patient is administered triamterene.

In some embodiments, a patient is administered a diuretic when the patient shows signs of heart failure. In some embodiments, a patient is administered a diuretic when the patient shows signs congestion. In some embodiments, a patient is administered a diuretic when the patient is at high risk of coronary artery disease (CAD). In some embodiments, a patient is administered a diuretic when the patient has coronary artery disease (CAD). In some embodiments, a diuretic delays onset of heart failure in a patient. In some embodiments, a diuretic prevents onset of heart failure in a patient. In some embodiments, administration of a diuretic increases length of life in a patient. In some embodiments, administration of a diuretic decreases length of a hospital stay in a patient. In some embodiments, administration of a diuretic prevents hospitalization of a patient. In some embodiments, administration of a diuretic improves a patient's six minute walk test.

Other

In some embodiments, a patient is administered one or more treatments selected from the group consisting of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine). In some embodiments a patient is administered hydralazine and isosorbide dinitrate. In some embodiments a patient is administered digoxin. In some embodiments a patient is administered digitalis. In some embodiments a patient is administered N-3 polyunsaturated fatty acids (PUFA). In some embodiments a patient is administered I_f-channel inhibitor (e.g., Ivabradine).

In some embodiments, a patient is administered one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) when the patient shows signs of heart failure. In some embodiments, one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) delays onset of heart failure in a patient. In some embodiments, one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) prevents onset of heart failure in a patient. In some embodiments, administration of one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) increases length of life in a patient. In some embodiments, administration of one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) decreases length of a hospital stay in a patient. In some embodiments, administration of one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) prevents hospitalization of a patient. In some embodiments, administration of one or more of hydralazine and isosorbide dinitrate, digoxin, digitalis, N-3 polyunsaturated fatty acids (PUFA), I_f-channel inhibitor (e.g., Ivabradine) improves a patient's six minute walk test.

9. Comorbidities

Comorbidities are important in HF and may affect the use of treatments for HF (e.g., it may not be possible to use renin-angiotensin system inhibitors in some patients with severe renal dysfunction). Furthermore, drugs used to treat comorbidities may cause worsening of HF (e.g., NSAIDs given for arthritis, some anti-cancer drugs, etc.). Therefore, management of comorbidities is a key component of the holistic care of patients with HF. In some embodiments, one or more comorbidities to consider in HF are selected from the group consisting of arterial hypertension, atrial fibrillation, cognitive dysfunction, diabetes, hypercholesterolemia, iron deficiency, kidney dysfunction, metabolic syndrome, obesity, physical deconditioning, potassium disorders, pulmonary disease (e.g., COPD), and sleep apnea.

In some embodiments, the disclosure contemplates methods of treating one or more comorbidities of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure contemplates methods of treating one or more comorbidities of heart failure (e.g., arterial hypertension, atrial fibrillation, cognitive dysfunction, diabetes, hypercholesterolemia, iron deficiency, kidney dysfunction, metabolic syndrome, obesity, physical deconditioning, potassium disorders, pulmonary disease (e.g., COPD), and sleep apnea) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the one or more comorbidities of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) are improved indirectly. In some embodiments, the disclosure contemplates methods of preventing one or more comorbidities of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure contemplates methods of reducing the progression rate of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure contemplates methods of reducing the progression rate of one or more comorbidities of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure contemplates methods of reducing the severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist). In some embodiments, the disclosure contemplates methods of reducing the severity of one or more comorbidities of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies) comprising administering to a patient in need thereof an effective amount of an ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist).

10. Screening Assays

In certain aspects, the present disclosure relates to the use of ActRII-ALK4 antagonist (e.g., an ActRII-ALK4 ligand trap antagonist, an ActRII-ALK4 antibody antagonist, an ActRII-ALK4 polynucleotide antagonist, and/or an ActRII-ALK4 small molecule antagonist) to identify compounds (agents) which may be used to treat, prevent, or reduce the progression rate and/or severity of heart failure (e.g., dilated cardiomyopathy (DCM), heart failure associated with muscle wasting diseases, and genetic cardiomyopathies), particularly treating, preventing or reducing the progression rate and/or severity of one or more heart failure-associated comorbidities.

There are numerous approaches to screening for therapeutic agents for treating heart failure by targeting signaling (e.g., Smad signaling) of one or more ActRII-ALK4 ligands. In certain embodiments, high-throughput screening of compounds can be carried out to identify agents that perturb ActRII-ALK4 ligands-mediated effects on a selected cell line. In certain embodiments, the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an ActRII-ALK4 ligand (e.g., activin A, activin B, activin AB, activin C, GDF3, BMP6, GDF8, GDF15, GDF11 or BMP10) to its binding partner, such as an a type II receptor (e.g., ActRIIA and/or ActRIIB). Alternatively, the assay can be used to identify compounds that enhance binding of an ActRII-ALK4 ligand to its binding partner such as a type II receptor. In a further embodiment, the compounds can be identified by their ability to interact with a type II receptor.

A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. As described herein, the test compounds (agents) of the invention may be created by any combinatorial chemical method. Alternatively, the subject compounds may be naturally occurring biomolecules synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. In certain embodiments, the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daltons.

The test compounds of the disclosure can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps. Optionally, the compounds may be optionally derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photoactivatable crosslinkers or any combinations thereof.

In many drug-screening programs which test libraries of compounds and natural extracts, high-throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity between an ActRII-ALK4 ligand (e.g., activin A, activin B, activin AB, activin C, GDF8, GDF15, GDF11, GDF3, BMP6, or BMP10) to its binding partner, such as an a type II receptor (e.g., ActRIIA and/or ActRIIB).

Merely to illustrate, in an exemplary screening assay of the present disclosure, the compound of interest is contacted with an isolated and purified ActRIIB polypeptide which is ordinarily capable of binding to an ActRIIB ligand, as appropriate for the intention of the assay. To the mixture of the compound and ActRIIB polypeptide is then added to a composition containing an ActRIIB ligand (e.g., GDF11). Detection and quantification of ActRIIB/ActRIIB-ligand complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the ActRIIB polypeptide and its binding protein. The efficacy of the compound can be assessed by generating dose-response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified ActRIIB ligand is added to a composition containing the ActRIIB polypeptide, and the formation of ActRIIB/ActRIIB ligand complex is quantitated in the absence of the test compound. It will be understood that, in general, the order in which the reactants may be admixed can be varied, and can be admixed simultaneously. Moreover, in place of purified proteins, cellular extracts and lysates may be used to render a suitable cell-free assay system.

Complex formation between an ActRII-ALK4 ligand and its binding protein may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIB polypeptide and/or its binding protein, by immunoassay, or by chromatographic detection.

In certain embodiments, the present disclosure contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between a GDF/BMP ligand and its binding protein. Further, other modes of detection, such as those based on optical waveguides (see, e.g., PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the disclosure.

Moreover, the present disclosure contemplates the use of an interaction trap assay, also known as the “two-hybrid assay,” for identifying agents that disrupt or potentiate interaction between an ActRII-ALK4 ligand and its binding partner. See, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment, the present disclosure contemplates the use of reverse two-hybrid systems to identify compounds (e.g., small molecules or peptides) that dissociate interactions between an ActRII-ALK4 ligand and its binding protein [see, e.g., Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and U.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368].

In certain embodiments, the subject compounds are identified by their ability to interact with an ActRII-ALK4 ligand. The interaction between the compound and the ActRII-ALK4 ligand may be covalent or non-covalent. For example, such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography [see, e.g., Jakoby W B et al. (1974) Methods in Enzymology 46:1]. In certain cases, the compounds may be screened in a mechanism-based assay, such as an assay to detect compounds which bind to an ActRII-ALK4 ligand. This may include a solid-phase or fluid-phase binding event. Alternatively, the gene encoding ActRII-ALK4 ligand can be transfected with a reporter system (e.g., 0-galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library preferably by high-throughput screening or with individual members of the library. Other mechanism-based binding assays may be used; for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound compounds may be detected usually using colorimetric endpoints or fluorescence or surface plasmon resonance.

11. Pharmaceutical Compositions

The therapeutic agents described herein (e.g., ActRII-ALK4 antagonists) may be formulated into pharmaceutical compositions. Pharmaceutical compositions for use in accordance with the present disclosure may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Such formulations will generally be substantially pyrogen-free, in compliance with most regulatory requirements.

In certain embodiments, the therapeutic methods of the disclosure include administering the composition systemically, or locally as an implant or device. When administered, the therapeutic composition for use in this disclosure is in a substantially pyrogen-free, or pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than the ActRII-ALK4 antagonists which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds in the methods disclosed herein.

Typically, protein therapeutic agents disclosed herein will be administered parentally, and particularly intravenously or subcutaneously. Pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRII-ALK4 antagonists in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions and formulations may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration

Further, the composition may be encapsulated or injected in a form for delivery to a target tissue site. In certain embodiments, compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., ActRII-ALK4 antagonists) to a target tissue site, providing a structure for the developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow release of the ActRII-ALK4 antagonist. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the subject compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.

In certain embodiments, methods of the invention can be administered for orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient. An agent may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

It is understood that the dosage regimen will be determined by the attending physician considering various factors which modify the action of the subject compounds of the disclosure (e.g., ActRII-ALK4 antagonists). The various factors include, but are not limited to, the patient's age, sex, and diet, the severity disease, time of administration, and other clinical factors. Optionally, the dosage may vary with the type of matrix used in the reconstitution and the types of compounds in the composition. The addition of other known growth factors to the final composition, may also affect the dosage. Progress can be monitored by periodic assessment of bone growth and/or repair, for example, X-rays (including DEXA), histomorphometric determinations, and tetracycline labeling.

In certain embodiments, the present invention also provides gene therapy for the in vivo production of ActRII-ALK4 antagonists. Such therapy would achieve its therapeutic effect by introduction of the ActRII-ALK4 antagonist polynucleotide sequences into cells or tissues having the disorders as listed above. Delivery of ActRII-ALK4 antagonist polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Preferred for therapeutic delivery of ActRII-ALK4 antagonist polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody. Those of skill in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ActRII-ALK4 antagonist. In a preferred embodiment, the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for ActRII-ALK4 antagonist polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using a liposome vehicle, are known in the art, see e.g., Mannino, et al., Biotechniques, 6:682, 1988. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art.

The disclosure provides formulations that may be varied to include acids and bases to adjust the pH; and buffering agents to keep the pH within a narrow range.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments of the present invention, and are not intended to limit the invention.

Example 1: ActRIIA-Fc Fusion Proteins

A soluble ActRIIA fusion protein was constructed that has the extracellular domain of human ActRIIA fused to a human or mouse Fc domain with a minimal linker in between. The constructs are referred to as ActRIIA-hFc and ActRIIA-mFc, respectively.

ActRIIA-hFc is shown below as purified from CHO cell lines (SEQ ID NO: 380):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
EVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An additional ActRIIA-hFc lacking the C-terminal lysine is shown below as purified from CHO cell lines (SEQ ID NO: 378):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
EVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The ActRIIA-hFc and ActRIIA-mFc proteins were expressed in CHO cell lines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML):
(SEQ ID NO: 7)
MKFLVNVALVFMVVYISYIYA
(ii) Tissue plasminogen activator (TPA):
(SEQ ID NO: 8)
MDAMKRGLCCVLLLCGAVFVSP
(iii) Native:
(SEQ ID NO: 379)
MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the following unprocessed amino acid sequence:

(SEQ ID NO: 381)
MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQT
GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKK
DSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 382)
ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGC
AGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGG
AGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTG
GTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCT
ACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGG
CTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGA
CAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAA
AGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCA
GTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC
AGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGAGAATTC

Both ActRIIA-hFc and ActRIIA-mFc were remarkably amenable to recombinant expression. As shown in FIG. 14, the protein was purified as a single, well-defined peak of protein. N-terminal sequencing revealed a single sequence of -ILGRSETQE (SEQ ID NO: 383). Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. The ActRIIA-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE.

ActRIIA-hFc and ActRIIA-mFc showed a high affinity for ligands. GDF11 or activin A were immobilized on a Biacore™ CM5 chip using standard amine-coupling procedure. ActRIIA-hFc and ActRIIA-mFc proteins were loaded onto the system, and binding was measured. ActRIIA-hFc bound to activin with a dissociation constant (K_D) of 5×10⁻¹² and bound to GDF11 with a K_D of 9.96×10⁻⁹. See FIG. 15A-B. Using a similar binding assay, ActRIIA-hFc was determined to have high to moderate affinity for other TGF-beta superfamily ligands including, for example, activin B, GDF8, BMP6, and BMP10. ActRIIA-mFc behaved similarly.

The ActRIIA-hFc was very stable in pharmacokinetic studies. Rats were dosed with 1 mg/kg, 3 mg/kg, or 10 mg/kg of ActRIIA-hFc protein, and plasma levels of the protein were measured at 24, 48, 72, 144 and 168 hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg, or 30 mg/kg. In rats, ActRIIA-hFc had an 11-14 day serum half-life, and circulating levels of the drug were quite high after two weeks (11 Ig/ml, 110 pg/ml, or 304 pg/ml for initial administrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasma half-life was substantially greater than 14 days, and circulating levels of the drug were 25 pg/ml, 304 pg/ml, or 1440 pg/ml for initial administrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively.

Example 2: Characterization of an ActRIIA-hFc Protein

ActRIIA-hFc fusion protein was expressed in stably transfected CHO-DUKX B 11 cells from a pAID4 vector (SV40 on/enhancer, CMV promoter), using a tissue plasminogen leader sequence of SEQ ID NO: 8. The protein, purified as described above in Example 1, had a sequence of SEQ ID NO: 380. The Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO: 380. Protein analysis reveals that the ActRIIA-hFc fusion protein is formed as a homodimer with disulfide bonding.

The CHO-cell-expressed material has a higher affinity for activin B ligand than that reported for an ActRIIA-hFc fusion protein expressed in human 293 cells [see, del Re et al. (2004) J Biol Chem. 279(51):53126-53135]. Additionally, the use of the TPA leader sequence provided greater production than other leader sequences and, unlike ActRIIA-Fc expressed with a native leader, provided a highly pure N-terminal sequence. Use of the native leader sequence resulted in two major species of ActRIIA-Fc, each having a different N-terminal sequence.

Example 3: Alternative ActRIIA-Fc Proteins

A variety of ActRIIA variants that may be used according to the methods described herein are described in the International Patent Application published as WO2006/012627 (see e.g., pp. 55-58), incorporated herein by reference in its entirety. An alternative construct may have a deletion of the C-terminal tail (the final 15 amino acids of the extracellular domain of ActRIIA. The sequence for such a construct is presented below (Fc portion underlined) (SEQ ID NO: 384):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 4. Generation of an ActRIIB-Fc Fusion Polypeptide

Applicants constructed a soluble ActRIIB fusion polypeptide that has the extracellular domain of human ActRIIB fused to a human G1Fc domain with a linker (three glycine amino acids) in between. The construct is referred to as ActRIIB(20-134)-G1Fc.

ActRIIB(20-134)-G1Fc is shown below in SEQ ID NO: 5 (with the linker underlined) as purified from CHO cell lines:

(SEQ ID NO: 5)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGT
IELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA
GGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An additional ActRIIB(20-134)-G1Fc lacking the C-terminal lysine is shown below as purified from CHO cell lines (SEQ ID NO: 378):

(SEQ ID NO: 385)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGT
IELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA
GGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The ActRIIB(20-134)-G1Fc polypeptide was expressed in CHO cell lines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML):
(SEQ ID NO: 7)
MKFLVNVALVFMVVYISYIYA
(ii) Tissue plasminogen activator (TPA):
(SEQ ID NO: 8)
MDAMKRGLCCVLLLCGAVFVSP
(iii) Native:
(SEQ ID NO: 9)
MTAPWVALALLWGSLCAG.

The selected form employs the TPA leader and has the following unprocessed amino acid sequence:

(SEQ ID NO: 6)
MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQS
GLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATE
ENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 10):

(SEQ ID NO: 10)
ATGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT
GTGTGGAGCA GTCTTCGTTT CGCCCGGCGC CTCTGGGCGT
GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCA
ACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG
CTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCC
TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGA
AGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA
GGAGTGTGTG GCCACTGAGG AGAACCCCCA GGTGTACTTC
TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTC
ATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC
ACCCCCGACA GCCCCCACCG GTGGTGGAAC TCACACATGC
CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAG
TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT
CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG
AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG
ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA
GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC
ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA
AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA
GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA
CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA
AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA
TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG
CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACT
CCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA
CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC
GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA
GCCTCTCCCT GTCTCCGGGT AAATGA

N-terminal sequencing of the CHO-cell produced material revealed a major sequence of -GRGEAE (SEQ ID NO: 11). Notably, other constructs reported in the literature begin with an -SGR . . . sequence.

Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange.

The ActRIIB(20-134)-Fc fusion polypeptide was also expressed in HEK293 cells and COS cells. Although material from all cell lines and reasonable culture conditions provided polypeptide with muscle-building activity in vivo, variability in potency was observed perhaps relating to cell line selection and/or culture conditions.

Example 5. Computational Methods

The Activin IIB receptor (ActRIIB) binds multiple TGFβ superfamily ligands, including activin A, activin B, GDF8, and GDF11, that stimulate Smad2/3 activation, as well as bone morphogenic proteins (BMPs), such as BMP9 and BMP10, that stimulate Smad1/5/8 activation. ActRIIB-Fc fusion polypeptides can function as ligand traps that bind to soluble ligands and block Smad activation by preventing ligands from binding to cell surface receptors. ActRIIB-Fc antagonism of BMP9-mediated Smad1/5/8 activation has been known to result in undesired side effects, including epistaxis and telangiectasias (Campbell, C. et al. Muscle Nerve 55: 458-464, 2017). In order to design mutations in ActRIIB that diminish BMP9 binding, while retaining binding to ligands that stimulate Smad2/3 activation, we compared the crystal structures of three ActRIIB ligand complexes: (1) BMP9:ActRIIB:Alk1, PDB ID=4fao, (2) ActRIIB:Activin A, PDB ID: 1s4y, and (3) GDF11:ActRIIB:Alk5, PDB ID: 6mac (available from the Protein Data Bank (PDB) https://www.rcsb.org/). Comparison of contacts between ActRIIB and the three ligands based on the crystal structures revealed residues for mutational focus based on charge, polarity, and hydrophobicity differences of the ligand residues contacted by the same corresponding ActRIIB residue. After identifying residues to target for mutation, the Schrödinger Bioluminate biologics modeling software platform (version 2017-4: Bioluminate, Schrödinger, LLC, New York, NY) was used to computationally predict mutations in ActRIIB that would diminish binding to BMP9, while maintaining other ligand-binding activities.

All residues identified from the comparison of the crystal structures were considered for mutation. Residue Scanning Calculations were performed considering both stability and affinity of the molecules in the structural complex, producing a specified list of potential mutations and energies for each molecule (ligand and receptor) and complex structure, as well as energy differences for both the wild type and the mutant form. After analyzing affinity/stability/prime energy, etc. parameters, the top 5%-10% of the single mutations were identified. This analysis was followed by potential combination of these mutations. Selected single mutations and mutation combinations were structurally analyzed in order to understand structural differences and formed/lost contacts. Ultimately, 817 single mutations were screened for each complex (ActRIIB:ligand), and top hits were selected based on Δaffinity, and also taking into selective consideration Δstability (solvated) and Δprime energy. Other properties were also considered when regarding striking of outliers.

Example 6. Generation of Variant ActRIIB-Fc Polypeptides

Based on the findings described in Example 4, Applicants generated a series of mutations (sequence variations) in the extracellular domain of ActRIIB and produced these variant polypeptides as soluble homodimeric fusion polypeptides comprising a variant ActRIIB extracellular domain and an Fc domain joined by an optional linker. The background ActRIIB-Fc fusion used for the generation of variant ActRIIB-Fc polypeptides was ActRIIB-G1Fc, and is shown in Example 4 above as SEQ ID NO: 5.

Various substitution mutations were introduced into the background ActRIIB-G1Fc polypeptide. Based on the data presented in Example 4, it is expected that these constructs, if expressed with a TPA leader, will lack the N-terminal serine. Thus, the majority of mature sequences may begin with a glycine (lacking the N-terminal serine) but some species may be present with the N-terminal serine. Mutations were generated in the ActRIIB extracellular domain by PCR mutagenesis. After PCR, fragments were purified through a Qiagen column, digested with SfoI and AgeI and gel purified. These fragments were ligated into expression vector pAID4 (see WO2006/012627) such that upon ligation it created fusion chimera with human IgG1. Upon transformation into E. coli DH5 alpha, colonies were picked and DNA was isolated. For murine constructs (mFc), a murine IgG2a was substituted for the human IgG1. All mutants were sequence verified.

The amino acid sequence of unprocessed ActRIIB(F82I-N83R)-G1Fc is shown below (SEQ ID NO: 276). The signal sequence and linker sequence are indicated by solid underline, and the F82I and N83R substitutions are indicated by double underline. The amino acid sequence of SEQ ID NO: 276 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 276)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA
    NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDIRC
    YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT
    GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE
    DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY
    KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV
    KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
    GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(F82I-N83R)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 277):

(SEQ ID NO: 277)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC
     TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT
     GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC
     CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC
     ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG
     AAGGGCTGCT
251  GGCTAGATGA CATCCGTTGC TACGATAGGC AGGAGTGTGT
     GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT
     TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC
     ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG
     CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT
     TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC
     ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA
     CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
     AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG
     CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA
     AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
     CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC
     AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA
     CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA
     CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG
     CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC
     CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
     TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(F82I-N83R)-G1Fc fusion polypeptide (SEQ ID NO: 278) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 278)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC
    YASWRNSSGT
51  IELVKKGCWL DDIRCYDRQE CVATEENPQV YFCCCEGNFC
    NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP
    PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE
    QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
    EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT
    PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
    PGK

The amino acid sequence of unprocessed ActRIIB(F82K-N83R)-G1Fc is shown below (SEQ ID NO: 279). The signal sequence and linker sequence are indicated by solid underline, and the F82K and N83R substitutions are indicated by double underline. The amino acid sequence of SEQ ID NO: 279 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 279)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA
    NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDKRC
    YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT
    GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE
    DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY
    KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV
    KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
    GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(F82K-N83R)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 331):

(SEQ ID NO: 331)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC
     TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT
     GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC
     CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC
     ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG
     AAGGGCTGCT
251  GGCTAGATGA CAAGCGTTGC TACGATAGGC AGGAGTGTGT
     GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT
     TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC
     ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG
     CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT
     TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC
     ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA
     CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
     AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG
     CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA
     AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
     CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC
     AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA
     CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA
     CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG
     CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC
     CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
     TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(F82K-N83R)-G1Fc fusion polypeptide (SEQ ID NO: 332) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 332)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC
    YASWRNSSGT
51  IELVKKGCWL DDKRCYDRQE CVATEENPQV YFCCCEGNFC
    NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP
    PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE
    QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
    EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT
    PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
    PGK

The amino acid sequence of unprocessed ActRIIB(F82T-N83R)-G1Fc is shown below (SEQ ID NO: 333). The signal sequence and linker sequence are indicated by solid underline, and the F82T and N83R substitutions are indicated by double underline. The amino acid sequence of SEQ ID NO: 333 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 333)
1   MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA
    NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDTRC
    YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT
    GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE
    DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY
    KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV
    KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
    GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(F82T-N83R)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 334):

(SEQ ID NO: 334)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC
     TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT
     GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC
     CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC
     ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG
     AAGGGCTGCT
251  GGCTAGATGA CACCCGTTGC TACGATAGGC AGGAGTGTGT
     GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT
     TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC
     ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG
     CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT
     TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC
     ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA
     CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
     AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG
     CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA
     AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
     CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC
     AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA
     CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA
     CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG
     CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC
     CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
     TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(F82T-N83R)-G1Fc fusion polypeptide (SEQ ID NO: 335) is as

(SEQ ID NO: 335)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC
    YASWRNSSGT
51  IELVKKGCWL DDTRCYDRQE CVATEENPQV YFCCCEGNFC
    NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP
    PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE
    QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
    EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT
    PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS
    PGK

The amino acid sequence of unprocessed ActRIIB(F82T)-G1Fc is shown below (SEQ ID NO: 336). The signal sequence and linker sequence are indicated by solid underline, and the F82T substitution is indicated by double underline. The amino acid sequence of SEQ ID NO: 336 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 336)
1   MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA
    NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDTNC
    YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT
    GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE
    DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY
    KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV
    KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
    GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(F82T)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 337):

(SEQ ID NO: 337)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC
     TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT
     GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC
     CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC
     ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG
     AAGGGCTGCT
251  GGCTAGATGA CACCAACTGC TACGATAGGC AGGAGTGTGT
     GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT
     TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC
     ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG
     CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT
     TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC
     ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA
     CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
     AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG
     CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA
     AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
     CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC
     AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA
     CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA
     CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG
     CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC
     CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
     TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(F82T)-G1Fc fusion polypeptide (SEQ ID NO: 338) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 338)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC
    YASWRNSSGT
51  IELVKKGCWL DDTNCYDRQE CVATEENPQV YFCCCEGNFC
    NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP
    PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE
    QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR
    EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT
    PPVLDSDGSE
301 FLYSKLTVDK SRWQQGNVES CSVMHEALHN HYTQKSLSLS
    PGK

The amino acid sequence of unprocessed ActRIIB(L79H-F82I)-G1Fc is shown below (SEQ ID NO: 339). The signal sequence and linker sequence are indicated by solid underline, and the L79H and F82I substitutions are indicated by double underline. The amino acid sequence of SEQ ID NO: 339 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 339)
1   MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA
    NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWHDDINC
    YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT
    GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE
    DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY
    KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV
    KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
    GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(L79H-F82I)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 340):

(SEQ ID NO: 340)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCACGATGA CATCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(L79H-F82I)-G1Fc fusion polypeptide (SEQ ID NO: 341) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 341)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWH DDINCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(L79H)-G1Fc is shown below (SEQ ID NO: 342). The signal sequence and linker sequence are indicated by solid underline, and the L79H substitution is indicated by double underline. The amino acid sequence of SEQ ID NO: 342 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 342)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWHDDFNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(L79H)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 343):

(SEQ ID NO: 343)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCACGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(L79H)-G1Fc fusion polypeptide (SEQ ID NO: 344) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 344)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWH DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(L79H-F82K)-G1Fc is shown below (SEQ ID NO: 345). The signal sequence and linker sequence are indicated by solid underline, and the L79H and F82K substitutions are indicated by double underline. The amino acid sequence of SEQ ID NO: 345 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 345)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWHDDKNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(L79H-F82K)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 346):

(SEQ ID NO: 346)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCACGATGA CAAGAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(L79H-F82K)-G1Fc fusion polypeptide (SEQ ID NO: 347) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 347)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWH DDKNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(E50L)-G1Fc is shown below (SEQ ID NO: 348). The signal sequence and linker sequence are indicated by solid underline, and the E50L substitution is indicated by double underline. The amino acid sequence of SEQ ID NO: 348 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 348)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCLGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVESCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(E50L)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (codon optimized) (SEQ ID NO: 349):

(SEQ ID NO: 349)
1    ATGGATGCGA TGAAACGCGG CCTGTGCTGC GTGCTGCTGC TGTGCGGCGC
51   GGTGTTTGTG AGCCCGGGCG CCAGCGGCCG CGGCGAAGCG GAAACCCGCG
101  AATGCATTTA TTATAACGCG AACTGGGAAC TGGAACGCAC CAACCAGAGC
151  GGCCTGGAAC GCTGCCTGGG CGAACAGGAT AAACGCCTGC ATTGCTATGC
201  GAGCTGGCGC AACAGCAGCG GCACCATTGA ACTGGTGAAA AAAGGCTGCT
251  GGCTGGATGA TTTTAACTGC TATGATCGCC AGGAATGCGT GGCGACCGAA
301  GAAAACCCGC AGGTGTATTT TTGCTGCTGC GAAGGCAACT TTTGCAACGA
351  ACGCTTTACC CATCTGCCGG AAGCGGGCGG CCCGGAAGTG ACCTATGAAC
401  CGCCGCCGAC CGCGCCGACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(E50L)-G1Fc fusion polypeptide (SEQ ID NO: 350) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 350)
1   GRGEAETREC IYYNANWELE RTNQSGLERC LGEQDKRLHC YASWRNSSGT
51  IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(L38N-L79R)-G1Fc is shown below (SEQ ID NO: 351). The signal sequence and linker sequence are indicated by solid underline, and the L38N and L79R substitutions are indicated by double underline. The amino acid sequence of SEQ ID NO: 351 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 351)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWENERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWRDDFNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(L38N-L79R)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 352):

(SEQ ID NO: 352)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGA ACGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCGCGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(L38N-L79R)-G1Fc fusion polypeptide (SEQ ID NO: 353) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 353)
1   GRGEAETREC IYYNANWENE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWR DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(V99G)-G1Fc is shown below (SEQ ID NO: 354). The signal sequence and linker sequence are indicated by solid underline, and the V99G substitution is indicated by double underline. The amino acid sequence of SEQ ID NO: 354 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 354)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE
101 ENPQGYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(V99G)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (codon optimized) (SEQ ID NO: 355):

(SEQ ID NO: 355)
1 ATGGATGCGA TGAAACGCGG CCTGTGCTGC GTGCTGCTGC TGTGCGGCGC
51 GGTGTTTGTG AGCCCGGGCG CCAGCGGCCG CGGCGAAGCG GAAACCCGCG
101 AATGCATTTA TTATAACGCG AACTGGGAAC TGGAACGCAC CAACCAGAGC
151 GGCCTGGAAC GCTGCGAAGG CGAACAGGAT AAACGCCTGC ATTGCTATGC
201 GAGCTGGCGC AACAGCAGCG GCACCATTGA ACTGGTGAAA AAAGGCTGCT
251 GGCTGGATGA TTTTAACTGC TATGATCGCC AGGAATGCGT GGCGACCGAA
301 GAAAACCCGC AGGGCTATTT TTGCTGCTGC GAAGGCAACT TTTGCAACGA
351 ACGCTTTACC CATCTGCCGG AAGCGGGCGG CCCGGAAGTG ACCTATGAAC
401 CGCCGCCGAC CGCGCCGACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951 CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAATGA

A mature ActRIIB(V99G)-G1Fc fusion polypeptide (SEQ ID NO: 356) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 356)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWL DDENCYDRQE CVATEENPQG YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Constructs were expressed in COS or CHO cells by transient infection and purified by filtration and protein A chromatography. In some instances, assays were performed with conditioned medium rather than purified polypeptides. Purity of samples for reporter gene assays was evaluated by SDS-PAGE and analytical size exclusion chromatography.

Mutants were tested in binding assays and/or bioassays described below.

Alternatively, similar mutations could be introduced into an ActRIIB extracellular domain possessing an N-terminal truncation of five amino acids and a C-terminal truncation of three amino acids as shown below (SEQ ID NO: 357). This truncated ActRIIB extracellular domain is denoted ActRIIB(25-131) based on numbering in SEQ ID NO: 2.

(SEQ ID NO: 357)
25 ETRECIYYNA NWELERTNQS GLERCEGEQD
KRLHCYASWR NSSGTIELVK
75 KGCWLDDFNC YDRQECVATE ENPQVYFCCC
EGNFCNERFT HLPEAGGPEV
125 TYEPPPT

The corresponding background fusion polypeptide, ActRIIB(25-131)-G1Fc, is shown below (SEQ ID NO: 12).

(SEQ ID NO: 12)
1 ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWR NSSGTIELVK
51 KGCWLDDENC YDRQECVATE ENPQVYFCCC EGNFCNERFT HLPEAGGPEV
101 TYEPPPTGGG THTCPPCPAP ELLGGPSVEL FPPKPKDTLM ISRTPEVTCV
151 VVDVSHEDPE VKENWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
201 WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
251 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
301 DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

Example 7. Activity and Ligand Binding Profiles of Variant ActRIIB-Fc Polypeptides

To determine ligand binding profiles of variant ActRIIB-Fc homodimers, a Biacore™-based binding assay was used to compare ligand binding kinetics of certain variant ActRIIB-Fc polypeptides. ActRIIB-Fc polypeptides to be tested were independently captured onto the system using an anti-Fc antibody. Ligands were then injected and allowed to flow over the captured receptor protein. Results of variant ActRIIB-Fc polypeptides analyzed at 37° C. are shown in FIGS. 16A and 16B. ActRIIB-G1Fc was used as the control polypeptide.

To determine activity of variant ActRIIB-Fc polypeptides, an A204 cell-based assay was used to compare effects among variant ActRIIB-Fc polypeptides on signaling by activin A, activin B, GDF8, GDF11, BMP9, and BMP10, in comparison to ActRIIB-G1Fc. In brief, this assay uses a human A204 rhabdomyosarcoma cell line (ATCC®: HTB-82™) derived from muscle and the reporter vector pGL3(CAGA)12 (Dennler et al., 1998, EMBO 17: 3091-3100) as well as a Renilla reporter plasmid (pRLCMV) to control for transfection efficiency. The CAGA12 motif is present in TGF-β responsive genes (e.g., PAI-1 gene), so this vector is of general use for ligands that can signal through Smad2/3, including activin A, GDF11, and BMP9.

On day 1, A204 cells were transferred into one or more 48-well plates. On day 2, these cells were transfected with 10 pg pGL3(CAGA)12 or pGL3(CAGA)12(10 pg)+pRLCMV (1 pg) and Fugene. On day 3, ligands diluted in medium containing 0.1% BSA were preincubated with ActRIIB-Fc polypeptides for 1 hr before addition to cells. Approximately six hour later, the cells were rinsed with PBS and lysed. Cell lysates were analyzed in a luciferase assay to determine the extent of Smad activation.

This assay was used to screen variant ActRIIB-Fc polypeptides for inhibitory effects on cell signaling by activin A, activin B, GDF8, GDF11, BMP9, and BMP10. Potencies of homodimeric Fc fusion polypeptides incorporating amino acid substitutions in the human ActRIIB extracellular domain were compared with that of an Fc fusion polypeptide comprising unmodified human ActRIIB extracellular domain, ActRIIB-G1Fc. For some variants tested, it was not possible to calculate an accurate IC₅₀, but signs of inhibition in the slope of the curves were detectable. For these variants, an estimate was included of the order of magnitude of the relative IC50, i.e. >10 nM or >100 nM instead of a definite number. Such data points are indicated by a (*) in Table 10 below. For some variants tested, there was no detectable inhibition in the slope of the curves over the concentration range tested, which is indicated by “ND” in Table 10.

TABLE 10
Inhibitory Potency of Homodimeric ActRIIB-Fc Constructs.
Inhibitory Potency of Homodimeric ActRIIB-Fc Constructs
ActRIIB	IC50 (nM)
poly-			Activin	Activin
peptide	GDF8	GDF11	A	B	BMP9	BMP10
ActRIIB-	0.95	0.12	0.05	0.067	1.82	0.036
G1Fc
F82I-N83R	ND	9.95	1.67	0.08	ND	13.25
F82K-N83R	ND	ND	1.32	0.09	ND	0.53
F82T-N83R	ND	17.94	1.52	0.11	ND	12.57
F82T	2.17	0.27	0.10	0.09	ND	0.07
L79H-F82I	>10*	0.36	>100*	0.15	ND	>100*
L79H	5.76	0.24	>10*	0.07	ND	>100*
L79H-F82K	ND	>100*	ND	0.10	ND	>100*
ND: not detectable over concentration range tested
*estimate of the order of magnitude of the IC50

As shown in Table 10 above as well as in FIGS. 16A and 16B, amino acid substitutions in the ActRIIB extracellular domain can alter the balance between ActRIIB:ligand binding and downstream signaling activities in various in vitro assay. In general, applicant achieved the goal of generating variants in the ActRIIB extracellular domain that exhibited decreased or non-detectable binding to BMP9, compared to a fusion polypeptide containing unmodified ActRIIB extracellular domain (ActRIIB-G1Fc), while retaining other ligand binding properties.

Additionally, variants ActRIIB (L79H-F82I), ActRIIB (L79H), and ActRIIB (L79H-F82K), while demonstrating a decrease in binding to BMP9, also exhibited a significant decrease in in activin A binding while retaining relatively high affinity for activin B, as compared to ActRIIB-G1Fc. IC₅₀ values showing inhibitory potency in Table 10 are consistent with this ligand binding trend. Similarly, variants ActRIIB (F82K-N83R), ActRIIB (F82I-N83R), and ActRIIB (F82T-N83R) demonstrate a similar trend.

Furthermore, variants ActRIIB (F82K-N83R), ActRIIB (F82I-N83R), ActRIIB (F82T-N83R), and ActRIIB (L79H-F82K), while demonstrating a decrease in binding to BMP9 and retaining relatively high affinity for activin B, also exhibited a significant decrease in GDF8 and GDF11 binding, as compared to ActRIIB-G1Fc. IC₅₀ values showing inhibitory potency in Table 10 are consistent with this ligand binding trend.

It was further noted that, variants ActRIIB (L79H-F82I), ActRIIB (L79H), and ActRIIB (L79H-F82K), while demonstrating a decrease in binding to BMP9 and retaining relatively high affinity for activin B, also exhibited a decrease in BMP10 binding as compared to ActRIIB-G1Fc. IC₅₀ values showing inhibitory potency in Table 10 are consistent with this ligand binding trend.

Therefore, in addition to achieving the goal of producing ActRIIB variants that exhibit reduced to non-detectable binding to BMP9, Applicant has generated a diverse array of novel variant polypeptides, many of which are characterized in part by unique ligand binding/inhibition profiles. Accordingly, these variants may be more useful than ActRIIB-G1Fc in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of activin B, while reducing antagonism of BMP9 and optionally one or more of activin A, GDF8, GDF11 and BMP10.

Example 8. Generation of Variant ActRIIB-Fc Polypeptides

Applicants generated a series of mutations (sequence variations) in the extracellular domain of ActRIIB and produced these variant polypeptides as soluble homodimeric fusion polypeptides comprising a variant ActRIIB extracellular domain and an Fc domain joined by an optional linker. The background ActRIIB-Fc fusion was ActRIIB-G1Fc as shown in SEQ ID NO: 5.

Various substitution mutations were introduced into the background ActRIIB-Fc polypeptide. Based on the data presented in Example 4, it is expected that these constructs, if expressed with a TPA leader, will lack the N-terminal serine. Mutations were generated in the ActRIIB extracellular domain by PCR mutagenesis. After PCR, fragments were purified through a Qiagen column, digested with SfoI and AgeI and gel purified. These fragments were ligated into expression vector pAID4 (see WO2006/012627) such that upon ligation it created fusion chimera with human IgG1. Upon transformation into E. coli DH5 alpha, colonies were picked and DNA was isolated. For murine constructs (mFc), a murine IgG2a was substituted for the human IgG1. All mutants were sequence verified.

The amino acid sequence of unprocessed ActRIIB(K55A)-G1Fc is shown below (SEQ ID NO: 31). The signal sequence and linker sequence are indicated by solid underline, and the K55A substitution is indicated by double underline. The amino acid sequence of SEQ ID NO:31 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 31)
1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD ARLHCYASWR NSSGTIELVK KGCWLDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(K55A)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 32):

(SEQ ID NO: 32)
1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC GCCCGGCTGC ACTGCTACGC
201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251 GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951 CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCCCCGGG
1101 TAAA

The mature ActRIIB(K55A)-G1Fc fusion polypeptide (SEQ ID NO: 33) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 33)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDARLHC YASWRNSSGT
51 IELVKKGCWL DDENCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(K55E)-G1Fc is shown below (SEQ ID NO: 34). The signal sequence and linker sequence are indicated by solid underline and the K55E substitution is indicated by double underline. The amino acid sequence of SEQ ID NO:34 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 34)
1 MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD ERLHCYASWR NSSGTIELVK KGCWLDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(K55E)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 35):

(SEQ ID NO: 35)
1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC GAGCGGCTGC ACTGCTACGC
201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251 GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951 CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCCCCGGG
1101 TAAA

The mature ActRIIB(K55E)-G1Fc fusion polypeptide (SEQ ID NO: 36) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 36)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDERLHC YASWRNSSGT
51 IELVKKGCWL DDENCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(F82I)-G1Fc is shown below (SEQ ID NO: 37). The signal sequence and linker sequence are indicated by solid underline and the F82I substitution is indicated by double underline. The amino acid sequence of SEQ ID NO: 37 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 37)
1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDINC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VELFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVESCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(F82I)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 38):

(SEQ ID NO: 38)
1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251 GGCTAGATGA CATCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951 CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAA

The mature ActRIIB(F82I)-G1Fc fusion polypeptide (SEQ ID NO: 39) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 39)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWL DDINCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVELFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The amino acid sequence of unprocessed ActRIIB(F82K)-G1Fc is shown below (SEQ ID NO: 40). The signal sequence and linker sequence are indicated by solid underline and the F82K substitution is indicated by double underline. The amino acid sequence of SEQ ID NO: 40 may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 40)
1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDKNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

This ActRIIB(F82K)-G1Fc fusion polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 41):

(SEQ ID NO: 41)
1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251 GGCTAGATGA CAAGAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951 CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAA

The mature ActRIIB(F82K)-G1Fc fusion polypeptide (SEQ ID NO: 42) is as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 42)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWL DDKNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVELFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVES CSVMHEALHN HYTQKSLSLS PGK

Constructs were expressed in COS or CHO cells and purified by filtration and protein A chromatography. In some instances, assays were performed with conditioned medium rather than purified proteins. Purity of samples for reporter gene assays was evaluated by SDS-PAGE and Western blot analysis.

Mutants were tested in binding assays and/or bioassays described below.

Alternatively, similar mutations could be introduced into an ActRIIB extracellular domain possessing an N-terminal truncation of five amino acids and a C-terminal truncation of three amino acids as shown below (SEQ ID NO: 53). This truncated ActRIIB extracellular domain is denoted ActRIIB(25-131) based on numbering in SEQ ID NO: 2.

(SEQ ID NO: 53)
25 ETRECIYYNA NWELERTNQS GLERCEGEQD
KRLHCYASWR NSSGTIELVK
75 KGCWLDDFNC YDRQECVATE ENPQVYFCCC
EGNFCNERFT HLPEAGGPEV
125 TYEPPPT

The corresponding background fusion polypeptide, ActRIIB(25-131)-G1Fc, is shown below (SEQ ID NO: 12).

(SEQ ID NO: 12)
1 ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWR NSSGTIELVK
51 KGCWLDDENC YDRQECVATE ENPQVYFCCC EGNFCNERFT HLPEAGGPEV
101 TYEPPPTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV
151 VVDVSHEDPE VKENWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD
201 WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ
251 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
301 DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

Example 9. Ligand Binding Profiles of Variant ActRIIB-Fc Homodimers and Activity of Variant ActRIIB-Fc Polypeptides in a Cell-Based Assay

To determine ligand binding profiles of variant ActRIIB-Fc homodimers, a Biacore™-based binding assay was used to compare ligand binding kinetics of certain variant ActRIIB-Fc polypeptides. ActRIIB-Fc polypeptides to be tested were independently captured onto the system using an anti-Fc antibody. Ligands were then injected and allowed to flow over the captured receptor protein. Results of variant ActRIIB-Fc polypeptides analyzed at 37° C. are shown in FIG. 17. Compared to Fc-fusion polypeptide comprising unmodified ActRIIB extracellular domain, the variant polypeptides ActRIIB(K55A)-Fc, ActRIIB(K55E)-Fc, ActRIIB(F82I)-Fc, and ActRIIB(F82K)-Fc exhibited greater reduction in their affinity for BMP9 than for GDF11. Results of additional variant ActRIIB-Fc polypeptides analyzed at 25° C. are shown in FIG. 18.

These results confirm K55A, K55E, F82I, and F82K as substitutions that reduce ActRIIB binding affinity for BMP9 more than they reduce ActRIIB affinity for activin A or GDF11. Accordingly, these variant ActRIIB-Fc polypeptides may be more useful than unmodified ActRIIB-Fc polypeptide in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of one or more of activin A, activin B, GDF8, and GDF11 while reducing antagonism of BMP9.

To determine activity of variant ActRIIB-Fc polypeptides, an A204 cell-based assay was used to compare effects among variant ActRIIB-Fc polypeptides on signaling by activin A, GDF11, and BMP9. In brief, this assay uses a human A204 rhabdomyosarcoma cell line (ATCC®: HTB-82™) derived from muscle and the reporter vector pGL3(CAGA)12 (Dennler et al., 1998, EMBO 17: 3091-3100) as well as a Renilla reporter plasmid (pRLCMV) to control for transfection efficiency. The CAGA12 motif is present in TGF-β responsive genes (e.g., PAI-1 gene), so this vector is of general use for ligands that can signal through Smad2/3, including activin A, GDF11, and BMP9.

On day 1, A-204 cells were transferred into one or more 48-well plates. On day 2, these cells were transfected with 10 pg pGL3(CAGA)12 or pGL3(CAGA)12(10 μg)+pRLCMV (1 μg) and Fugene. On day 3, ligands diluted in medium containing 0.1% BSA were preincubated with ActRIIB-Fc polypeptides for 1 hr before addition to cells. Approximately six hour later, the cells were rinsed with PBS and lysed. Cell lysates were analyzed in a luciferase assay to determine the extent of Smad activation.

This assay was used to screen variant ActRIIB-Fc polypeptides for inhibitory effects on cell signaling by activin A, GDF11, and BMP9. Potencies of homodimeric Fc fusion polypeptides incorporating amino acid substitutions in the human ActRIIB extracellular domain were compared with that of an Fc fusion polypeptide comprising unmodified human ActRIIB extracellular domain.

TABLE 11
Inhibitory Potency of Homodimeric
ActRIIB-Fc Constructs
	ActRIIB	IC50 (ng/ml)
	polypeptide	Activin A	GDF11	BMP9
	Wild-type	8	9	31
	A24N	128	99	409
	R40A	—	591	1210
	E50K	132	180	721
	E50P	756	638	~3000
	E52A	198	71	359
	E52K	762	296	~10000
	K55A	15	11	122
	K55D	396	365	5500
	K55E	19	14	290
	K55R	206	318	777
	Y60K	—	414	ND
	Y60P	—	544	ND
	K74R	—	45	165
	K74Y	—	ND	ND
	K74A/L79P	—	ND	ND
	L79K	—	477	ND
	L79P	—	ND	ND
	L79R	—	234	ND
	D80A	—	ND	ND
	F82I	11	9	277
	F82K	10	15	~5000
	F82W	—	276	ND
	F82W/N83A	—	389	~40000
	V99E	—	ND	ND
	V99K	—	ND	—
	ND: not detectable over concentration range tested
	—Not tested

As shown in the table above, single amino acid substitutions in the ActRIIB extracellular domain can alter the balance between activin A or GDF11 inhibition and BMP9 inhibition in a cell-based reporter gene assay. Compared to a fusion polypeptide containing unmodified ActRIIB extracellular domain, the variants ActRIIB(K55A)-Fc, ActRIIB(K55E)-Fc, ActRIIB(F82I)-Fc, and ActRIIB(F82K)-Fc showed less potent inhibition of BMP9 (increased IC₅₀ values) while maintaining essentially undiminished inhibition of activin A and GDF11.

These results indicate that variant ActRIIB-Fc polypeptides such as ActRIIB(K55A)-Fc, ActRIIB(K55E)-Fc, ActRIIB(F82I)-Fc, and ActRIIB(F82K)-Fc are more selective antagonists of activin A and GDF11 compared to an Fc fusion polypeptide comprising unmodified ActRIIB extracellular domain. Accordingly, these variants may be more useful than ActRIIB-Fc in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of one or more of activin A, GDF8, and GDF11 while reducing antagonism of BMP9 and potentially BMP10.

Example 10. Generation of an ActRIIB-Fc:ActRIIB(L79E)-Fc Heterodimer

Applicants envision generation of a soluble ActRIIB-Fc:ActRIIB(L79E)-Fc heteromeric complex comprising the extracellular domains of unmodified human ActRIIB and human ActRIIB with a leucine-to-glutamate substitution at position 79, which are each separately fused to an G1Fc domain with a linker positioned between the extracellular domain and the G1Fc domain. The individual constructs are referred to as ActRIIB-Fc fusion polypeptide and ActRIIB(L79E)-Fc fusion polypeptide, respectively, and the sequences for each are provided below.

A methodology for promoting formation of ActRIIB-Fc:ActRIIB(L79E)-Fc heteromeric complexes, as opposed to the ActRIIB-Fc or ActRIIB(L79E)-Fc homodimeric complexes, is to introduce alterations in the amino acid sequence of the Fc domains to guide the formation of asymmetric heteromeric complexes. Many different approaches to making asymmetric interaction pairs using Fc domains are described in this disclosure.

In one approach, illustrated in the ActRIIB(L79E)-Fc and ActRIIB-Fc polypeptide sequences of SEQ ID NOs: 43-45 and 46-48, respectively, one Fc domain can be altered to introduce cationic amino acids at the interaction face, while the other Fc domain can be altered to introduce anionic amino acids at the interaction face. The ActRIIB(L79E)-Fc fusion polypeptide and ActRIIB-Fc fusion polypeptide can each employ the TPA leader (SEQ ID NO: 8).

The ActRIIB(L79E)-Fc polypeptide sequence (SEQ ID NO: 43) is shown below:

(SEQ ID NO: 43)
1 MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWEDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYDTTPPVLD SDGSFFLYSD LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPG

The leader (signal) sequence and linker are underlined, and the L79E substitution is indicated by double underline. To promote formation of the ActRIIB-Fc:ActRIIB(L79E)-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing lysines with acidic amino acids) can be introduced into the Fc domain of the ActRIIB fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 43 may optionally be provided with lysine added to the C-terminus.

This ActRIIB(L79E)-Fc fusion polypeptide can be encoded by the following nucleic acid sequence (SEQ ID NO: 44):

(SEQ ID NO: 44)
1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251 GGGAAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACGACA CCACGCCTCC
951 CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCGAC CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 T

The mature ActRIIB(L79E)-Fc fusion polypeptide (SEQ ID NO: 45) is as follows, and may optionally be provided with lysine added to the C-terminus.

(SEQ ID NO: 45)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWE DDENCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYDTT PPVLDSDGSF
301 FLYSDLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PG

The complementary form of ActRIIB-Fc fusion polypeptide (SEQ ID NO: 46) is as follows:

(SEQ ID NO: 46)
1 MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VELFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWINGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

The leader sequence and linker sequence are underlined. To guide heterodimer formation with the ActRIIB(L79E)-Fc fusion polypeptide of SEQ ID NOs: 43 and 45 above, two amino acid substitutions (replacing a glutamate and an aspartate with lysines) can be introduced into the Fc domain of the ActRIIB-Fc fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 46 may optionally be provided with lysine removed from the C-terminus.

This ActRIIB-Fc fusion polypeptide can be encoded by the following nucleic acid (SEQ ID NO: 47):

(SEQ ID NO: 47)
1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101 AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151 GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201 CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251 GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401 CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801 ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG
851 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951 CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAA

The mature ActRIIB-Fc fusion polypeptide sequence (SEQ ID NO: 48) is as follows and may optionally be provided with lysine removed from the C-terminus:

(SEQ ID NO: 48)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWL DDENCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLKSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The ActRIIB(L79E)-Fc and ActRIIB-Fc polypeptides of SEQ ID NO: 45 and SEQ ID NO: 48, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric polypeptide complex comprising ActRIIB-Fc:ActRIIB(L79E)-Fc.

In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion polypeptides, the Fc domains can be altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond as illustrated in the ActRIIB(L79E)-Fc and ActRIIB-Fc polypeptide sequences of SEQ ID NOs: 49-50 and 51-52, respectively. The ActRIIB(L79E)-Fc fusion polypeptide and ActRIIB-Fc fusion polypeptide can each employ the TPA leader (SEQ ID NO: 8). ActRIIB(L79E)-Fc polypeptide sequence (SEQ ID NO: 49) is shown below:

(SEQ ID NO: 49)
1 MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWEDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPG

The signal sequence and linker sequence are underlined, and the L79E substitution is indicated by double underline. To promote formation of the ActRIIB-Fc:ActRIIB(L79E)-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a tryptophan) can be introduced into the Fc domain of the fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 49 may optionally be provided with lysine added to the C-terminus. Mature ActRIIB(L79E)-Fc fusion polypeptide (SEQ ID NO: 50) is as follows:

(SEQ ID NO: 50)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWE DDENCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVELFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC
251 REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PG

The complementary form of ActRIIB-Fc fusion polypeptide (SEQ ID NO: 51) is as follows and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 51)
1 MDAMKRGLCC VLLLCGAVEV SPGASGRGEA ETRECIYYNA NWELERTNQS
51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VELFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKENWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVC TLPPSREEMT KNQVSLSCAV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVESCSVMH
351 EALHNHYTQK SLSLSPGK

The leader sequence and linker are underlined. To guide heterodimer formation with the ActRIIB(L79E)-Fc fusion polypeptide of SEQ ID NOs: 49-50 above, four amino acid substitutions (replacement of tyrosine with cysteine, threonine with serine, leucine with alanine, and tyrosine with valine) can be introduced into the Fc domain of the ActRIIB-Fc fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 51 may optionally be provided with lysine removed from the C-terminus.

The mature ActRIIB-Fc fusion polypeptide sequence is as follows and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 52)
1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51 IELVKKGCWL DDENCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVCTLPPS
251 REEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLVSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The ActRIIB(L79E)-Fc and ActRIIB-Fc polypeptides of SEQ ID NO: 50 and SEQ ID NO: 52, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric polypeptide complex comprising ActRIIB-Fc:ActRIIB(L79E)-Fc.

Purification of various ActRIIB-Fc:ActRIIB(L79E)-Fc complexes can be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, cation exchange chromatography, multimodal chromatography (e.g., with resin containing both electrostatic and hydrophobic ligands), and epitope-based affinity chromatography (e.g., with an antibody or functionally equivalent ligand directed against an epitope of ActRIIB). The purification can be completed with viral filtration and buffer exchange.

Example 11. Ligand Binding Profile of ActRIIB-Fc:ActRIIB(L79E)-Fc Heteromer

A Biacore™-based binding assay was used to compare the ligand binding kinetics of an ActRIIB-Fc:ActRIIB(L79E)-Fc heterodimer with those of unmodified ActRIIB-Fc homodimer. Fusion proteins were captured onto the system using an anti-Fc antibody. Ligands were then injected and allowed to flow over the captured receptor protein at 37° C. Results are summarized in the table below, in which ligand off-rates (k_d) most indicative of effective ligand traps are denoted in bold.

TABLE 12
Ligand binding of ActRIIB-Fc:ActRIIB(L79E)-Fc heterodimer
compared to ActRII-Fc homodimer at 37° C.
	ActRIIB-Fc	ActRIIB-Fc:ActRIIB(L79E)-Fc
	homodimer	heterodimer
	ka	kd	KD	ka	kd	KD
Ligand	(1/Ms)	(1/s)	(pM)	(1/Ms)	(1/s)	(pM)
Activin	7.4 × 106	1.9 × 10 −4	25	8.8 × 106	1.5 × 10−3	170
A
Activin	8.1 × 106	6.6 × 10 −5	8	8.3 × 106	2.1 × 10 −4	25
B
GDF3	1.4 × 106	2.2 × 10−3	1500	5.8 × 105	5.9 × 10−3	10000
GDF8	3.8 × 106	2.6 × 10 −4	70	3.4 × 106	5.0 × 10 −4	150
GDF11	4.1 × 107	1.7 × 10 −4	4	4.0 × 107	3.6 × 10 −4	9
BMP6	1.3 × 108	7.4 × 10−3	56	3.3 × 108	1.8 × 10−2	56
BMP9	5.0 × 106	1.3 × 10−3	250	Transient*	>2800
BMP10	5.1 × 107	2.0 × 10 −4	4	4.8 × 107	2.0 × 10−3	42
*Indeterminate due to transient nature of interaction

In this example, a single amino acid substitution in one of two ActRIIB polypeptide chains altered ligand binding selectivity of the Fc-fusion polypeptide relative to unmodified ActRIIB-Fc homodimer. Compared to ActRIIB-Fc homodimer, the ActRIIB(L79E)-Fc heterodimer largely retained high-affinity binding to activin B, GDF8, GDF11, and BMP6 but exhibited approximately ten-fold faster off-rates for activin A and BMP10 and an even greater reduction in the strength of binding to BMP9. Accordingly, a variant ActRIIB-Fc heteromer may be more useful than unmodified ActRIIB-Fc homodimer in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of one or more of activin B, GDF8, GDF11, and BMP6, while reducing antagonism of activin A, BMP9, or BMP10.9. Generation of ActRIIB mutants:

A series of mutations in the extracellular domain of ActRIIB were generated and these mutant polypeptides were produced as soluble fusion polypeptides between extracellular ActRIIB and an Fc domain. A co-crystal structure of Activin and extracellular ActRIIB did not show any role for the final (C-terminal) 15 amino acids (referred to as the “tail” herein) of the extracellular domain in ligand binding. This sequence failed to resolve on the crystal structure, suggesting that these residues are present in a flexible loop that did not pack uniformly in the crystal. Thompson EMBO J. 2003 Apr. 1; 22(7):1555-66. This sequence is also poorly conserved between ActRIIB and ActRIIA. Accordingly, these residues were omitted in the basic, or background, ActRIIB-Fc fusion construct. Additionally, in this example position 64 in the background form is occupied by an alanine. Thus, the background ActRIIB-Fc fusion in this example has the sequence (Fc portion underlined)(SEQ ID NO: 54):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASW
ANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNF
CNERFTHLPEAGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK

Surprisingly, as discussed below, the C-terminal tail was found to enhance activin and GDF-11 binding, thus a preferred version of ActRIIB-Fc has a sequence (Fc portion underlined)(SEQ ID NO: 55):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASW
ANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNF
CNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK

Various mutations were introduced into the background ActRIIB-Fc polypeptide. Mutations were generated in ActRIIB extracellular domain by PCR mutagenesis. After PCR, fragments were purified thru Qiagen column, digested with SfoI and AgeI and gel purified. These fragments were ligated into expression vector pAID4 such that upon ligation it created fusion chimera with human IgG1. DNAs were isolated. All of the mutants were produced in HEK293T cells by transient transfection. In summary, in a 500 ml spinner, HEK293T cells were set up at 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume and grown overnight. Next day, these cells were treated with DNA:PEI (1:1) complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml media was added and cells were grown for 7 days. Conditioned media was harvested by spinning down the cells and concentrated.

All the mutants were purified over protein A column and eluted with low pH (3.0) glycine buffer. After neutralization, these were dialyzed against PBS.

Mutants were also produced in CHO cells by similar methodology.

Mutants were tested in binding assays and bioassays described below. Proteins expressed in CHO cells and HEK293 cells were indistinguishable in the binding assays and bioassays.

Example 12: Generation of an ActRIIB-ALK4 Heterodimer

An ActRIIB-Fc:ALK4-Fc heteromeric complex was constructed comprising the extracellular domains of human ActRIIB and human ALK4, which are each separately fused to an Fc domain with a linker positioned between the extracellular domain and the Fc domain. The individual constructs are referred to as ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide, respectively, and the sequences for each are provided below.

A methodology for promoting formation of ActRIIB-Fc:ALK4-Fc heteromeric complexes, as opposed to ActRIIB-Fc or ALK4-Fc homodimeric complexes, is to introduce alterations in the amino acid sequence of the Fc domains to guide the formation of asymmetric heteromeric complexes. Many different approaches to making asymmetric interaction pairs using Fc domains are described in this disclosure.

In one approach, illustrated in the ActRIIB-Fc and ALK4-Fc polypeptide sequences of SEQ ID NOs: 396 and 398 and SEQ ID Nos: 88 and 89, respectively, one Fc domain is altered to introduce cationic amino acids at the interaction face, while the other Fc domain is altered to introduce anionic amino acids at the interaction face. ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader.

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 396) is shown below:

(SEQ ID NO: 396)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promote formation of ActRIIB-Fc:ALK4-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing acidic amino acids with lysine) can be introduced into the Fc domain of the ActRIIB fusion protein as indicated by double underline above. The amino acid sequence of SEQ ID NO: 396 may optionally be provided with lysine (K) removed from the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acid sequence (SEQ ID NO: 397):

(SEQ ID NO: 397)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAA

A mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 398) is as follows, and may optionally be provided with lysine (K) removed from the C-terminus.

(SEQ ID NO: 398)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLKSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

A complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 88) is as follows:

(SEQ ID NO: 88)
1   MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD
51  GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD
101 YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF
151 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
251 QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
301 DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VESCSVMHEA LHNHYTQKSL
351 SLSPG

The leader sequence and linker are underlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 396 and 398 above, two amino acid substitutions (replacing lysines with aspartic acids) can be introduced into the Fc domain of the ALK4-Fc fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 88 may optionally be provided with lysine (K) added at the C-terminus.

This ALK4-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO: 243):

(SEQ ID NO: 243)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC
101  TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT
151  GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT
201  GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT
251  ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC
301  TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC
351  TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA
401  CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC
451  CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA
501  GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT
551  TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG
601  CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT
651  CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA
701  ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG
751  CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT
801  GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA
851  GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC
901  GACACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCTATAG
951  CGACCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT
1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC
1051 TCCCTGTCTC CGGGT

A mature ALK4-Fc fusion protein sequence (SEQ ID NO: 89) is as follows and may optionally be provided with lysine (K) added at the C-terminus.

(SEQ ID NO: 89)
1   SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV
51  ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG
101 PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD
151 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
201 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
251 TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFF LYSDLTVDKS
301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

The ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 398 and SEQ ID NO: 89, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ActRIIB-Fc:ALK4-Fc.

In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion proteins the Fc domains are altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond as illustrated in the ActRIIB-Fc and ALK4-Fc polypeptide sequences of SEQ ID NOs: 402 and 403 and SEQ ID Nos: 92 and 93, respectively. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader: MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 8).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 402) is shown below:

(SEQ ID NO: 402)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promote formation of the ActRIIB-Fc:ALK4-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a tryptophan) can be introduced into the Fc domain of the fusion protein as indicated by double underline above. The amino acid sequence of SEQ ID NO: 402 may optionally be provided with lysine (K) removed from the C-terminus.

A mature ActRIIB-Fc fusion polypeptide is as follows:

(SEQ ID NO: 403)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC
251 REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

A complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 92) is as follows and may optionally be provided with lysine (K) removed from the C-terminus.

(SEQ ID NO: 92)
1   MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD
51  GACMVSIfNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD
101 YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF
151 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
251 QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESNGQPENNY
301 KTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL
351 SLSPGK

The leader sequence and the linker are underlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 402 and 403 above, four amino acid substitutions can be introduced into the Fc domain of the ALK4 fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 92 may optionally be provided with lysine (K) removed from the C-terminus.

A mature ALK4-Fc fusion protein sequence is as follows and may optionally be provided with lysine (K) removed from the C-terminus.

(SEQ ID NO: 93)
1   SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV
51  ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG
101 PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD
151 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
201 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL
251 SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS
301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 403 and SEQ ID NO: 93 respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ActRIIB-Fc:ALK4-Fc.

Purification of various ActRIIB-Fc:ALK4-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange.

In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion proteins, the Fc domains are altered to introduce complementary hydrophobic interactions, an additional intermolecular disulfide bond, and electrostatic differences between the two Fc domains for facilitating purification based on net molecular charge, as illustrated in the ActRIIB-Fc and ALK4-Fc polypeptide sequences of SEQ ID NOs: 118-121 and 122-125, respectively. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 406) is shown below:

(SEQ ID NO: 406)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPCREEMT ENQVSLWCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQD SLSLSPG

The leader sequence and linker are underlined. To promote formation of the ActRIIB-Fc:ALK4-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a tryptophan) can be introduced into the Fc domain of the fusion protein as indicated by double underline above. To facilitate purification of the ActRIIB-Fc:ALK4-Fcheterodimer, two amino acid substitutions (replacing lysines with acidic amino acids) can also be introduced into the Fc domain of the fusion protein as indicated by double underline above. The amino acid sequence of SEQ ID NO: 118 may optionally be provided with a lysine added at the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO: 407):

(SEQ ID NO: 407)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATGCCGGGA GGAGATGACC GAGAACCAGG
851  TCAGCCTGTG GTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGGAC AGCCTCTCCC TGTCTCCGGG
1101 T

The mature ActRIIB-Fc fusion polypeptide is as follows (SEQ ID NO: 408) and may optionally be provided with a lysine added to the C-terminus.

(SEQ ID NO: 408)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC
251 REEMTENQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVES CSVMHEALHN HYTQDSLSLS PG

This ActRIIB-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 409):

(SEQ ID NO: 409)
1    GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG
51   GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC
101  AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC
151  ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA
201  TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT
251  GCTGTGAAGG CAACTTCTGC AACGAGCGCT TCACTCATTT GCCAGAGGCT
301  GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACCGGTGG
351  TGGAACTCAC ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGGGGAC
401  CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC
451  CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC
501  TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA
551  AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG TGTGGTCAGC
601  GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG AGTACAAGTG
651  CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA
701  AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATGC
751  CGGGAGGAGA TGACCGAGAA CCAGGTCAGC CTGTGGTGCC TGGTCAAAGG
801  CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG
851  AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA CGGCTCCTTC
901  TTCCTCTATA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA
951  CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC
1001 AGGACAGCCT CTCCCTGTCT CCGGGT

The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 247) is as follows and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 247)
1   MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD
51  GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD
101 YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVE
151 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKENWYVDG VEVHNAKTKP
201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
251 QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESRGQPENNY
301 KTTPPVLDSR GSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL
351 SLSPGK

The leader sequence and the linker are underlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 406 and 408 above, four amino acid substitutions (replacing a tyrosine with a cysteine, a threonine with a serine, a leucine with an alanine, and a tyrosine with a valine) can be introduced into the Fc domain of the ALK4 fusion polypeptide as indicated by double underline above. To facilitate purification of the ActRIIB-Fc:ALK4-Fc heterodimer, two amino acid substitutions (replacing an asparagine with an arginine and an aspartate with an arginine) can also be introduced into the Fc domain of the ALK4-Fc fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 247 may optionally be provided with lysine removed from the C-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 248):

(SEQ ID NO: 248)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC
101  TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT
151  GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT
201  GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT
251  ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC
301  TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC
351  TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA
401  CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC
451  CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA
501  GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT
551  TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG
601  CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT
651  CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA
701  ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG
751  CAGCCCCGAG AACCACAGGT GTGCACCCTG CCCCCATCCC GGGAGGAGAT
801  GACCAAGAAC CAGGTCAGCC TGTCCTGCGC CGTCAAAGGC TTCTATCCCA
851  GCGACATCGC CGTGGAGTGG GAGAGCCGCG GGCAGCCGGA GAACAACTAC
901  AAGACCACGC CTCCCGTGCT GGACTCCCGC GGCTCCTTCT TCCTCGTGAG
951  CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT
1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC
1051 TCCCTGTCTC CGGGTAAA

The mature ALK4-Fc fusion polypeptide sequence is as follows (SEQ ID NO: 249) and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 249)
1   SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV
51  ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG
101 PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD
151 VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
201 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL
251 SCAVKGFYPS DIAVEWESRG QPENNYKTTP PVLDSRGSFF LVSKLTVDKS
301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO 250):

(SEQ ID NO: 250)
1   TCCGGGCCCC GGGGGGTCCA GGCTCTGCTG TGTGCGTGCA CCAGCTGCCT
51  CCAGGCCAAC TACACGTGTG AGACAGATGG GGCCTGCATG GTTTCCATTT
101 TCAATCTGGA TGGGATGGAG CACCATGTGC GCACCTGCAT CCCCAAAGTG
151 GAGCTGGTCC CTGCCGGGAA GCCCTTCTAC TGCCTGAGCT CGGAGGACCT
201 GCGCAACACC CACTGCTGCT ACACTGACTA CTGCAACAGG ATCGACTTGA
251 GGGTGCCCAG TGGTCACCTC AAGGAGCCTG AGCACCCGTC CATGTGGGGC
301 CCGGTGGAGA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC
351 TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG
401 ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC
451 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT
501 GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA
551 CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT
601 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT
651 CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT
701 GCACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG
751 TCCTGCGCCG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA
801 GAGCCGCGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG
851 ACTCCCGCGG CTCCTTCTTC CTCGTGAGCA AGCTCACCGT GGACAAGAGC
901 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT
951 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA

ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 120 and SEQ ID NO: 249, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

In certain embodiments, the ALK4-Fc fusion polypeptide is SEQ ID NO: 92 (shown above), which contains four amino acid substitutions to guide heterodimer formation certain Fc fusion polypeptides disclosed herein, and may optionally be provided with lysine removed from the C-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 251):

(SEQ ID NO: 251)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC
101  TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT
151  GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT
201  GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT
251  ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC
301  TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC
351  TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA
401  CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC
451  CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA
501  GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT
551  TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG
601  CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT
651  CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA
701  ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG
751  CAGCCCCGAG AACCACAGGT GTGCACCCTG CCCCCATCCC GGGAGGAGAT
801  GACCAAGAAC CAGGTCAGCC TGTCCTGCGC CGTCAAAGGC TTCTATCCCA
851  GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC
901  AAGACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCGTGAG
951  CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT
1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC
1051 TCCCTGTCTC CGGGTAAA

The mature ALK4-Fc fusion polypeptide sequence is SEQ ID NO: 93 (shown above) and may optionally be provided with lysine removed from the C-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid (SEQ ID NO: 252):

(SEQ ID NO: 252)
1   TCCGGGCCCC GGGGGGTCCA GGCTCTGCTG TGTGCGTGCA CCAGCTGCCT
51  CCAGGCCAAC TACACGTGTG AGACAGATGG GGCCTGCATG GTTTCCATTT
101 TCAATCTGGA TGGGATGGAG CACCATGTGC GCACCTGCAT CCCCAAAGTG
151 GAGCTGGTCC CTGCCGGGAA GCCCTTCTAC TGCCTGAGCT CGGAGGACCT
201 GCGCAACACC CACTGCTGCT ACACTGACTA CTGCAACAGG ATCGACTTGA
251 GGGTGCCCAG TGGTCACCTC AAGGAGCCTG AGCACCCGTC CATGTGGGGC
301 CCGGTGGAGA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC
351 TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG
401 ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC
451 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT
501 GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA
551 CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT
601 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT
651 CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT
701 GCACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG
751 TCCTGCGCCG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA
801 GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG
851 ACTCCGACGG CTCCTTCTTC CTCGTGAGCA AGCTCACCGT GGACAAGAGC
901 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT
951 GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA

Purification of various ActRIIB-Fc:ALK4-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, cation exchange chromatography, epitope-based affinity chromatography (e.g., with an antibody or functionally equivalent ligand directed against an epitope on ALK4 or ActRIIB), and multimodal chromatography (e.g., with resin containing both electrostatic and hydrophobic ligands). The purification could be completed with viral filtration and buffer exchange.

Example 13. Ligand Binding Profile of ActRIIB-Fc:ALK4-Fc Heterodimer Compared to ActRIIB-Fc Homodimer and ALK4-Fc Homodimer

A Biacore™-based binding assay was used to compare ligand binding selectivity of the ActRIIB-Fc:ALK4-Fc heterodimeric complex described above with that of ActRIIB-Fc and ALK4-Fc homodimer complexes. The ActRIIB-Fc:ALK4-Fc heterodimer, ActRIIB-Fc homodimer, and ALK4-Fc homodimer were independently captured onto the system using an anti-Fc antibody. Ligands were injected and allowed to flow over the captured receptor protein. Results are summarized in the table below, in which ligand off-rates (k_d) most indicative of effective ligand traps are denoted in bold.

Ligand binding profile of ActRIIB-Fc:ALK4-Fc heterodimer
compared to ActRIIB-Fc homodimer and ALK4-Fc homodimer
	ActRIIB-Fc	ALK4-Fc	ActRIIB-Fc:ALK4-Fc
	homodimer	homodimer	heterodimer
	ka	kd	KD	ka	kd	KD	ka	kd	KD
Ligand	(1/Ms)	(1/s)	(pM)	(1/Ms)	(1/s)	(pM)	(1/Ms)	(1/s)	(pM)
Activin	1.2 × 107	2.3 × 10 −4	19	5.8 × 105	1.2 × 10−2	20000	1.3 × 107	1.5 × 10 −4	12
A
Activin	5.1 × 106	1.0 × 10 −4	20	No binding	7.1 × 106	4.0 × 10 5	6
B
BMP6	3.2 × 107	6.8 × 103	190	—	2.0 × 106	5.5 × 10−3	2700
BMP9	1.4 × 107	1.1 × 103	77	—	Transient*	3400
BMP10	2.3 × 107	2.6 × 10 4	11	—	5.6 × 107	4.1 × 10−3	74
GDF3	1.4 × 106	2.2 × 103	1500	—	3.4 × 106	1.7 × 102	4900
GDF8	8.3 × 105	2.3 × 10 −4	280	1.3 × 105	1.9 × 10−3	15000†	3.9 × 105	2.1 × 10 −4	550
GDF11	5.0 × 107	1.1 × 10 −4	2	5.0 × 106	4.8 × 10−3	270†	3.8 × 107	1.1 × 10 4	3
*Indeterminate due to transient nature of interaction
†Very low signal
— Not tested

These comparative binding data demonstrate that ActRIIB-Fc:ALK4-Fc heterodimer has an altered binding profile/selectivity relative to either ActRIIB-Fc or ALK4-Fc homodimers. ActRIIB-Fc:ALK4-Fc heterodimer displays enhanced binding to activin B compared with either homodimer, retains strong binding to activin A, GDF8, and GDF11 as observed with ActRIIB-Fc homodimer, and exhibits substantially reduced binding to BMP9, BMP10, and GDF3. In particular, BMP9 displays low or no observable affinity for ActRIIB-Fc:ALK4-Fc heterodimer, whereas this ligand binds strongly to ActRIIB-Fc homodimer. Like the ActRIIB-Fc homodimer, the heterodimer retains intermediate-level binding to BMP6. See FIG. 19.

In addition, an A-204 Reporter Gene Assay was used to evaluate the effects of ActRIIB-Fc:ALK4-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimer on signaling by activin A, activin B, GDF11, GDF8, BMP10, and BMP9. Cell line: Human Rhabdomyosarcoma (derived from muscle). Reporter vector: pGL3(CAGA)12 (as described in Dennler et al, 1998, EMBO 17: 3091-3100). The CAGA12 motif is present in TGFβ responsive genes (PAI-1 gene), so this vector is of general use for factors signaling through Smad2 and 3. An exemplary A-204 Reporter Gene Assay is outlined below.

• • Day 1: Split A-204 cells into 48-well plate.
  • Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or pGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.
  • Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to be pre-incubated with Factors for about one hr before adding to cells. About six hrs later, cells are rinsed with PBS and then lysed.

Following the above steps, a Luciferase assay was performed.

Both the ActRIIB-Fc:ALK4-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimer were determined to be potent inhibitors of activin A, activin B, GDF11, and GDF8 in this assay. In particular, as can be seen in the comparative homodimer/heterodimer IC₅₀ data illustrated in FIG. 20, ActRIIB-Fc:ALK4-Fc heterodimer inhibits activin A, activin B, GDF8, and GDF11 signaling pathways similarly to the ActRIIB-Fc:ActRIIB-Fc homodimer. However, ActRIIB-Fc:ALK4-Fc heterodimer inhibition of BMP9 and BMP10 signaling pathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fc homodimer. This data is consistent with the above-discussed binding data in which it was observed that both the ActRIIB-Fc:ALK4-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimer display strong binding to activin A, activin B, GDF8, and GDF11, but BMP10 and BMP9 have significantly reduced affinity for the ALK4-Fc:ActRIIB-Fc heterodimer compared to the ActRIIB-Fc:ActRIIB-Fc homodimer.

Together, these data therefore demonstrate that ActRIIB-Fc:ALK4-Fc heterodimer is a more selective antagonist of activin A, activin B, GDF8, and GDF11 compared to ActRIIB-Fc homodimer. Accordingly, an ActRIIB-Fc:ALK4-Fc heterodimer will be more useful than an ActRIIB-Fc homodimer in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of one or more of activin A, activin B, activin AC, GDF8, and GDF11 but minimize antagonism of one or more of BMP9, BMP10, GDF3, and BMP6.

Example 14. Generation of an ActRIIB-Fc:ALK7-Fc Heterodimer

Applicants constructed a soluble ActRIIB-Fc:ALK7-Fc heteromeric complex comprising the extracellular domains of human ActRIIB and human ALK7, which are each fused to an Fc domain with a linker positioned between the extracellular domain and the Fc domain. The individual constructs are referred to as ActRIIB-Fc and ALK7-Fc, respectively.

A methodology for promoting formation of ActRIIB-Fc:ALK7-Fc heteromeric complexes, as opposed to the ActRIIB-Fc or ALK7-Fc homodimeric complexes, is to introduce alterations in the amino acid sequence of the Fc domains to guide the formation of asymmetric heteromeric complexes. Many different approaches to making asymmetric interaction pairs using Fc domains are described in this disclosure.

In one approach, illustrated in the ActRIIB-Fc and ALK7-Fc polypeptide sequences disclosed below, respectively, one Fc domain is altered to introduce cationic amino acids at the interaction face, while the other Fc domain is altered to introduce anionic amino acids at the interaction face. The ActRIIB-Fc fusion polypeptide and ALK7-Fc fusion polypeptide each employ the tissue plasminogen activator (TPA) leader: MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 8).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 396) is shown below:

(SEQ ID NO: 396)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDENC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promote formation of the ActRIIB-Fc:ALK7-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing acidic amino acids with lysine) can be introduced into the Fc domain of the ActRIIB fusion protein as indicated by double underline above. The amino acid sequence of SEQ ID NO: 396 may optionally be provided with lysine (K) removed from the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acid sequence (SEQ ID NO: 397):

(SEQ ID NO: 397)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG
101  AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC
151  GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC
201  CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT
251  GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG
301  GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA
351  GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC
401  CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC
451  CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA
501  ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
551  TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG
601  GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA
651  CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
701  GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA
751  GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC
801  ACAGGTGTAC ACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG
851  TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG
901  GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC
951  CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG
1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT
1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG
1101 TAAA

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 398) is as follows, and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 398)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
251 RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLKSDGSF
301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The complementary form of ALK7-Fc fusion protein (SEQ ID NO: 129) is as follows:

(SEQ ID NO: 129)
1   MDAMKRGLCC VLLLCGAVFV SPGAGLKCVC LLCDSSNFTC QTEGACWASV
51  MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VTKTECCFTD FCNNITLHLP
101 TASPNAPKLG PMETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR
151 TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
201 LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
251 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFF
301 LYSDLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

The signal sequence and linker sequence are underlined. To promote formation of the ActRIIB-Fc:ALK7-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing lysines with aspartic acids) can be introduced into the Fc domain of the fusion protein as indicated by double underline above. The amino acid sequence of SEQ ID NO: 129 may optionally be provided with a lysine added at the C-terminus.

This ALK7-Fc fusion protein is encoded by the following nucleic acid (SEQ ID NO: 255):

(SEQ ID NO: 255)
1    ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC
51   AGTCTTCGTT TCGCCCGGCG CCGGACTGAA GTGTGTATGT CTTTTGTGTG
101  ATTCTTCAAA CTTTACCTGC CAAACAGAAG GAGCATGTTG GGCATCAGTC
151  ATGCTAACCA ATGGAAAAGA GCAGGTGATC AAATCCTGTG TCTCCCTTCC
201  AGAACTGAAT GCTCAAGTCT TCTGTCATAG TTCCAACAAT GTTACCAAAA
251  CCGAATGCTG CTTCACAGAT TTTTGCAACA ACATAACACT GCACCTTCCA
301  ACAGCATCAC CAAATGCCCC AAAACTTGGA CCCATGGAGA CCGGTGGTGG
351  AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT
401  CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG
451  ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA
501  GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA
551  CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC
601  CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA
651  GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG
701  CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG
751  GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT
801  CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA
851  ACAACTACGA CACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC
901  CTCTATAGCG ACCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT
951  CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA
1001 AGAGCCTCTC CCTGTCTCCG GGT

The mature ALK7-Fc fusion protein sequence (SEQ ID NO: 130) is expected to be as follows and may optionally be provided with a lysine added at the C-terminus.

(SEQ ID NO: 130)
1   GLKCVCLLCD SSNFTCQTEG ACWASVMLIN GKEQVIKSCV SLPELNAQVF
51  CHSSNNVTKT ECCFTDFCNN ITLHLPTASP NAPKLGPMET GGGTHTCPPC
101 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
151 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
201 APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV
251 EWESNGQPEN NYDTTPPVLD SDGSFFLYSD LTVDKSRWQQ GNVESCSVMH
301 EALHNHYTQK SLSLSPG

The ActRIIB-Fc and ALK7-Fc fusion proteins of SEQ ID NO: 396 and SEQ ID NO: 129, respectively, may be co-expressed and purified from a CHO cell line to give rise to a heteromeric complex comprising ActRIIB-Fc:ALK7-Fc.

In another approach to promote the formation of heteromultimer complexes using asymmetric Fc fusion proteins, the Fc domains are altered to introduce complementary hydrophobic interactions and an additional intermolecular disulfide bond as illustrated in the ActRIIB-Fc and ALK7-Fc polypeptide sequences of disclosed below.

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 402) is shown below:

(SEQ ID NO: 402)
1   MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS
51  GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE
101 ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC
151 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
201 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP
251 APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV
301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVESCSVMH
351 EALHNHYTQK SLSLSPGK

The leader sequence and linker are underlined. To promote formation of the ActRIIB-Fc:ALK7-Fc heterodimer rather than either of the possible homodimeric complexes, two amino acid substitutions (replacing a serine with a cysteine and a threonine with a tryptophan) can be introduced into the Fc domain of the fusion protein as indicated by double underline above. The amino acid sequence of SEQ ID NO: 402 may optionally be provided with lysine removed from the C-terminus.

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 403) is as follows and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 403)
1   GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT
51  IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA
101 GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS
151 RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS
201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC
251 REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
301 FLYSKLTVDK SRWQQGNVES CSVMHEALHN HYTQKSLSLS PGK

The complementary form of ALK7-Fc fusion polypeptide (SEQ ID NO: 133) is as follows:

(SEQ ID NO: 133)
1   MDAMKRGLCC VLLLCGAVFV SPGAGLKCVC LLCDSSNFTC QTEGACWASV
51  MLTNGKEQVI KSCVSLPELN AQVFCHSSNN VIKTECCFTD FCNNITLHLP
101 TASPNAPKLG PMETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR
151 TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
201 LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR
251 EEMTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF
301 LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

The leader sequence and linker sequence are underlined. To guide heterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs 130 and 403 above, four amino acid substitutions can be introduced into the Fc domain of the ALK7 fusion polypeptide as indicated by double underline above. The amino acid sequence of SEQ ID NO: 133 may optionally be provided with the lysine removed from the C-terminus.

The mature ALK7-Fc fusion protein sequence (SEQ ID NO: 134) is expected to be as follows and may optionally be provided with the lysine removed from the C-terminus.

(SEQ ID NO: 134)
1   GLKCVCLLCD SSNFTCQTEG ACWASVMLTN GKEQVIKSCV SLPELNAQVF
51  CHSSNNVTKT ECCFTDFCNN ITLHLPTASP NAPKLGPMET GGGTHTCPPC
101 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
151 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWINGKEY KCKVSNKALP
201 APIEKTISKA KGQPREPQVC TLPPSREEMT KNQVSLSCAV KGFYPSDIAV
251 EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH
301 EALHNHYTQK SLSLSPGK

The ActRIIB-Fc and ALK7-Fc proteins of SEQ ID NO: 402 and SEQ ID NO: 133, respectively, may be co-expressed and purified from a CHO cell line, to give rise to a heteromeric complex comprising ActRIIB-Fc:ALK7-Fc.

Purification of various ActRIIB-Fc:ALK7-Fc complexes could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange.

Example 15. Ligand Binding Profile of ActRIIB-Fc:ALK7-Fc Heterodimer Compared to ActRIIB-Fc Homodimer and ALK7-Fc Homodimer

A Biacore™-based binding assay was used to compare ligand binding selectivity of the ActRIIB-Fc:ALK7-Fc heterodimeric complex described above with that of ActRIIB-Fc and ALK7-Fc homodimeric complexes. The ActRIIB-Fc:ALK7-Fc heterodimer, ActRIIB-Fc homodimer, and ALK7-Fc homodimer were independently captured onto the system using an anti-Fc antibody. Ligands were injected and allowed to flow over the captured receptor protein. Results are summarized in the table below, in which ligand off-rates (k_d) most indicative of effective ligand traps are denoted by in bold.

Ligand binding profile of ActRIIB-Fc:ALK7-Fc heterodimer
compared to ActRIIB-Fc homodimer and ALK7-Fc homodimer
	ActRIIB-Fc	ALK7-Fc	ActRIIB-Fc:ALK7-Fc
	homodimer	homodimer	heterodimer
	ka	kd	KD	ka	kd	KD	ka	kd	KD
Ligand	(1/Ms)	(1/s)	(pM)	(1/Ms)	(1/s)	(pM)	(1/Ms)	(1/s)	(pM)
activin A	1.3 × 107	1.4 × 10 −4	11	No binding	4.4 × 107	1.9 × 10−3	43
activin B	1.5 × 107	1.6 × 10 −4	8	No binding	1.2 × 107	2.0 × 10 −4	17
activin C	No binding	No binding	3.5 × 105	2.4 × 10−3	6900
activin	2.0 × 107	3.1 × 10−3	160	No binding	2.6 × 106	5.7 × 10 −4	220
AC
BMP5	2.6 × 107	7.5 × 10−2	2900	No binding	1.5 × 105	8.5 × 10−3	57000
BMP6	2.4 × 107	3.9 × 10−3	160	No binding	1.2 × 106	6.3 × 10−3	5300
BMP9	1.2 × 108	1.2 × 10−3	10	No binding	Transient*	>1400
BMP10	5.9 × 106	1.5 × 10 −4	25	No binding	1.5 × 107	2.8 × 10−3	190
GDF3	1.4 × 106	2.2 × 10−3	1500	No binding	2.3 × 106	1.0 × 10−2	4500
GDF8	3.5 × 106	2.4 × 10 −4	69	No binding	3.7 × 106	1.0 × 10−3	270
GDF11	9.6 × 107	1.5 × 10 −4	2	No binding	9.5 × 107	7.5 × 10 −4	8
*Indeterminate due to transient nature of interaction
— Not tested

These comparative binding data demonstrate that the ActRIIB-Fc:ALK7-Fc heterodimer has an altered binding profile/selectivity relative to either the ActRIIB-Fc homodimer or ALK7-Fc homodimer. Interestingly, four of the five ligands with the strongest binding to ActRIIB-Fc homodimer (activin A, BMP10, GDF8, and GDF11) exhibit reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, the exception being activin B which retains tight binding to the heterodimer. Similarly, three of the four ligands with intermediate binding to ActRIIB-Fc homodimer (GDF3, BMP6, and particularly BMP9) exhibit reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, whereas binding to activin AC is increased to become the second strongest ligand interaction with the heterodimer overall. Finally, activin C and BMP5 unexpectedly bind the ActRIIB-Fc:ALK7 heterodimer with intermediate strength despite no binding (activin C) or weak binding (BMP5) to ActRIIB-Fc homodimer. The net result is that the ActRIIB-Fc:ALK7-Fc heterodimer possesses a ligand-binding profile distinctly different from that of either ActRIIB-Fc homodimer or ALK7-Fc homodimer, which binds none of the foregoing ligands. See FIG. 21.

These results therefore demonstrate that the ActRIIB-Fc:ALK7-Fc heterodimer is a more selective antagonist of activin B and activin AC compared to ActRIIB-Fc homodimer. Moreover, ActRIIB-Fc:ALK7-Fc heterodimer exhibits the unusual property of robust binding to activin C. Accordingly, an ActRIIB-Fc:ALK7-Fc heterodimer will be more useful than an ActRIIB-Fc homodimer in certain applications where such selective antagonism is advantageous. Examples include therapeutic applications where it is desirable to retain antagonism of activin B or activin AC but decrease antagonism of one or more of activin A, GDF3, GDF8, GDF11, BMP9, or BMP10. Also included are therapeutic, diagnostic, or analytic applications in which it is desirable to antagonize activin C or, based on the similarity between activin C and activin E, activin E.

Example 16: The Role of ActRIIB-Fc:ALK4-Fc on Cardio-Protection in Heart Failure with Reduced Ejection Fraction (HFrEF)

Effects of ActRIIB-Fc:ALK4-Fc on cardio-protection were examined in a murine model of HFrEF: a transgenic, dystrophin-deficient mouse model called Mdx. Aged Mdx mice present typical phenotypes of dilated cardiomyopathy (e.g., phenotypes of HFrEF), including dilated left ventricular (LV) chamber, and eccentric hypertrophy of LV with relative wall thinning (FIG. 23A), accompanied by distinct LV systolic dysfunction (See, Arbustini, et al., Journal of the American College of Cardiology 2018, 72(20):2485-2506; Kamdar, et al., Journal of the American College of Cardiology 2016, 67(21):2533-2546; Houser, et al., Circulation Research 2012, 111(1):131-150; and Wasala, et al., American Society of Gene & Cell Therapy 2019, 28(3):845-854). Studies using Mdx mice were conducted to assess if ActRIIB-Fc:ALK4-Fc was able to restore cardiac morphological and functional alterations under remodeling.

Twenty-one Mdx male mice at 10-months of age (“Mid-age Mdx”) and 20-months of age (“Old Mdx”) were studied. Twelve age-matched wild type (WT) mice were included as a control (“Mid-age WT” and “Old WT”). Furthermore, three 3.5-month old WT male mice were used as an aging control, “Young WT”. “Mid-age Mdx” mice received either (i) vehicle (phosphate-buffered saline, PBS) twice per week subcutaneously for 6 months, or (ii) ActRIIB-Fc:ALK4-Fc (10 mg/kg) twice per week subcutaneously for 6 months. The volume of vehicle or ActRIIB-Fc:ALK4-Fc administered was the same. “Old Mdx” mice received either (i) vehicle (PBS) twice per week subcutaneously for 2 months, or (ii) ActRIIB-Fc:ALK4-Fc (10 mg/kg) twice per week subcutaneously for 2 months. The volume of vehicle or ActRIIB-Fc:ALK4-Fc administered was the same. All WT mice except for “Young WT” received the same administered dose of vehicle as its corresponding age-matched Mdx group. At the end of the study, before animals were euthanized, in vivo cardiac structure and function were assessed by transthoracic echocardiography (VisualSonics Vevo3100, 30 MHz transducer; Fujifilm) while mice were under anesthesia. Specifically, LV structure and systolic function were measured by M-mode in a parasternal short axis view at the papillary muscle level. Both LV wall thickness (LVWT) and LV mass (LVM) were obtained. LV end diastolic diameter (LVEDD) and LV end systolic diameter (LVESD) were measured and used to calculate fractional shortening (FS) using the following equation FS=100%×[(EDD−ESD)/EDD]. LV end diastolic volume (LVEDV) and LV end systolic volume (LVESV) were measured and used to calculate ejection fraction using the following equation EF=100%×[(EDV−ESV)/EDV]. Hypertrophy index was calculated as the ratio of LVM to LVESV. Relative wall thickness was calculated as the ratio of LVWT to LVESD. Right after echocardiography, all mice were euthanized, and their hearts were weighed. Blood of each mouse was collected and serum cardiac Troponin I expression was measured via high sensitivity ELISA.

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 or Person's correlation) were performed, with a significance level set as p<0.05. In particular, *p<0.05, **p<0.01, ***p<0.001.

By the end of the study, both Mid-age Mdx mice and Old Mdx mice displayed characteristic features of dilated cardiomyopathy, such as LV chamber dilation and systolic dysfunction. These cardiac morphological (FIG. 23) and functional (FIG. 24) deficits were completely restored by ActRIIB-Fc:ALK4-Fc treatment with either a short-term (e.g., 2 months of administration in Old-age Mdx mice) or a long-term (e.g., 6 months of administration in Mid-age Mdx mice) dosing regimen.

In particular, Mid-age Mdx-Vehicle and Old Mdx-Vehicle mice presented increase of left ventricular volume at the end of systole (FIG. 23B) compared to Young WT mice, meaning that less blood was ejected out of LV to aorta or to the rest of the body. Strikingly, LVESV in both Mid-age Mdx-ActRIIB-Fc:ALK4-Fc mice and Old Mdx-ActRIIB-Fc:ALK4-Fc mice was significantly reduced compared to Mid-age Mdx-Vehicle and Old Mdx-Vehicle groups, respectively, indicating that ActRIIB-Fc:ALK4-Fc improved LV contractility.

It was observed that LV remodeling of both Mid-age Mdx-Vehicle and Old Mdx-Vehicle mice underwent eccentric hypertrophy, with reduced ratio of mass to volume (i.e., hypertrophy index, FIG. 23C) compared to Young WT mice. The hypertrophy index was normalized by ActRIIB-Fc:ALK4-Fc treatment.

Accompanied by LV dilation and eccentric hypertrophy, LV wall thickness of Mid-age Mdx-Vehicle and Old Mdx-Vehicle mice was decreased compared to Young WT mice, shown in FIG. 23D, while ActRIIB-Fc:ALK4-Fc treatment increased relative LV wall thickness in both Mid-age Mdx-ActRIIB-Fc:ALK4-Fc mice and Old Mdx-ActRIIB-Fc:ALK4-Fc mice.

Eccentric hypertrophied LV, together with a relative thinning heart wall, also induced hypertrophied heart as shown in FIG. 23E. Normalized whole heart weight of Old Mdx-Vehicle mice was significantly increased compared to Young WT mice. ActRIIB-Fc:ALK4-Fc treatment as evidenced in Old Mdx-ActRIIB-Fc:ALK4-Fc mice, reduced heart weight.

These structural modifications under cardiac remodeling ensured cardiac functional alterations. Both Mid-age Mdx-Vehicle mice and Old Mdx-Vehicle mice displayed impaired contractility as evidenced by reduced ejection fraction (FIG. 24A) and fractional shortening (FIG. 24B) compared to Young WT mice. Strikingly, ActRIIB-Fc:ALK4-Fc treatment fully restored systolic function in both Mid-age Mdx-ActRIIB-Fc:ALK4-Fc mice and Old Mdx-ActRIIB-Fc:ALK4-Fc mice. In addition, elevated serum cardiac Troponin I (i.e., cTnI, a serum biomarker of cardiac injury) level was found at higher levels in Mid-age Mdx-Vehicle mice compared to Young WT mice. ActRIIB-Fc:ALK4-Fc treatment substantially decreased serum cTnI expression (See Mid-age Mdx-ActRIIB-Fc:ALK4-Fc mice and Old Mdx-ActRIIB-Fc:ALK4-Fc, FIG. 24C). Moreover, an inverse correlation between ejection fraction and cTnI was found, as seen in FIG. 24D, indicating that improved LV contractility by ActRIIB-Fc:ALK4-Fc may result from rescuing myocardium injury.

Together, these data demonstrate that ActRIIB-Fc:ALK4-Fc is effective to ameliorate various morphological and functional deficits during left heart remodeling in a murine model of HFrEF (Mdx model). In particular, LV end systolic diameter was significantly reduced in ActRIIB-Fc:ALK4-Fc treated mice compared to untreated groups, indicating that ActRIIB-Fc:ALK4-Fc improved LV contractility. The data further suggest that, in addition to ActRIIB:ALK4 heteromultimers, other ActRII-ALK4 antagonists may be useful in treating heart failure.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1-299. (canceled)

300. A method of treating heart failure (HF) in a patient in need thereof, comprising administering to the patient an effective amount of an ActRIIB-ALK4 antagonist.

301. The method of claim 300, wherein the ActRIIB-ALK4 antagonist is a heteromultimer comprising an ActRIIB polypeptide and an ALK4 polypeptide.

302. The method of claim 301, wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 2 or wherein the ActRIIB polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 25-131 of SEQ ID NO: 2.

303. The method of claim 301, wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 84 or wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 86.

304. The method of claim 301, wherein the heteromultimer comprises an ActRIIB fusion polypeptide comprising an ActRIIB polypeptide domain and one or more heterologous domains and/or an ALK4 fusion polypeptide comprising an ALK4 polypeptide domain and one or more heterologous domains.

305. The method of claim 304, wherein the ActRIIB or ALK4 fusion polypeptide further comprises a linker domain positioned between the polypeptide domain and the one or more heterologous domains selected from: TGGG, TGGGG, SGGGG, GGGGS, GGG, GGGG, SGGG, and GGGGS.

306. The method of claim 304, wherein the one or more heterologous domains is an Fc domain.

307. The method of claim 304, wherein the ALK4 fusion polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 92 or SEQ ID NO: 93.

308. The method of claim 304, wherein the ActRIIB fusion polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 402 or SEQ ID NO: 403

309. The method of claim 300, wherein the heart failure is selected from genetic cardiomyopathy, dilated cardiomyopathy (DCM) or heart failure with preserved ejection fraction (HFpEF).

310. The method of claim 300, wherein the patient has; i. normal left ventricular ejection fraction (LVEF) and an LVEF of ≥50%;ii. reduced LVEF and an LVEF of <40%;iii. mid-range LVEF and an LVEF of between about 40% and about 49%;iv diastolic dysfunction of the left ventricle (LV); orv. systolic dysfunction of the left ventricle (LV).

311. The method of claim 300, wherein the patient has elevated levels of natriuretic peptides.

312. The method of claim 300, wherein the heart failure is heart failure with mid-range ejection fraction (HFmrEF) or reduced ejection fraction (HFrEF).

313. The method of claim 300, wherein the method decreases troponin levels in the patient by at least 1%.

314. The method of claim 300, wherein the method decreases left ventricular hypertrophy in the patient by at least 1%.

315. The method of claim 300, wherein the method increases the patient's LV diastolic function by at least 5%.

316. The method of claim 300, wherein the method increases ejection fraction in the patient by at least 1%.

317. The method of claim 300, wherein the method increases the patient's cardiac output by at least 5%.

318. A method of treating heart failure (HF) in a patient in need thereof, comprising administering to the patient an effective amount of an ActRIIB-ALK4 heteromultimer, wherein the heteromultimer comprises (1) an ActRIIB fusion polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 402 or SEQ ID NO: 403; and(2) an ALK4 fusion polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 92 or SEQ ID NO: 93.

319. A method of treating, preventing, or reducing the progression rate and/or severity of one or more comorbidities of heart failure, comprising administering to a patient in need thereof an effective amount of an ActRIIB-ALK4 antagonist.