Compositions and method for treating kidney disease

Abstract

To provide activin and / or GDF antagonists for treating kidney disease, and to provide methods of using activin and / or GDF antagonists to treat kidney disease.SOLUTION: Provided is a method of treating or preventing kidney disease in a subject, comprising administering to the subject an activin and / or growth and differentiation factor (GDF) antagonist, the antagonist inhibiting one or more of activin, GDF8, GDF11, GDF3, GDF1, nodal, activin receptor type IIA (ActRIIA), ActRIIB, activin receptor-like kinase 4 (ALK4), ALK5, ALK7, Crypto-1, Cryptic, Cryptic 1B, Smad2, and Smad3.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 404,603, filed October 5, 2016, the entire contents of which are incorporated herein by reference in their entirety.

[0002] In some aspects, the disclosure relates to activin and / or GDF antagonists and methods of using activin and / or GDF antagonists to treat, prevent, or reduce the rate of progression and / or severity of kidney disease, particularly to treat, prevent, or reduce the rate of progression and / or severity of one or more kidney disease-related complications. [Background technology]

[0003] Kidney disease includes a range of conditions that can cause loss of kidney function and, in some cases, can be fatal. Normally functioning kidneys filter waste products and excess fluid from the blood, which are then excreted in urine. When chronic kidney disease reaches an advanced stage, dangerous levels of fluid, electrolytes and waste products can build up in the blood. If left untreated, kidney disease can progress to end-stage renal failure, which is fatal unless artificial filtration (dialysis) or kidney transplantation is performed. Thus, there is a high unmet need for effective therapies to treat kidney disease. Summary of the Invention

[0004] In some aspects, the disclosure relates to a method of treating kidney disease or kidney-related disease or disorder, comprising administering an effective amount of an activin and / or GDF antagonist (inhibitor), or a combination of activin and / or GDF antagonists (inhibitors) to a patient in need thereof. In certain aspects, the disclosure relates to a method of reducing the rate of progression of kidney disease, comprising administering an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonists to a patient in need thereof. In certain aspects, the disclosure relates to a method of reducing the severity of kidney disease, comprising administering an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonists to a patient in need thereof. In certain aspects, the present disclosure relates to a method of reducing the frequency of kidney-related disease events (e.g., kidney tissue damage, fibrosis and / or inflammation), comprising administering to a patient in need thereof an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonists. In certain aspects, the present disclosure relates to a method of treating one or more complications of kidney disease (e.g., kidney tissue damage, fibrosis and / or inflammation), comprising administering to a patient in need thereof an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonists. In certain aspects, the present disclosure relates to a method of preventing one or more complications of kidney disease, comprising administering to a patient in need thereof an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonists. In certain aspects, the present disclosure relates to methods of reducing the rate of progression of one or more complications of kidney disease comprising administering to a patient in need thereof an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonists.In certain aspects, the present disclosure relates to a method for reducing the severity of one or more complications of kidney disease, comprising administering an effective amount of an activin and / or GDF antagonist, or a combination of activin and / or GDF antagonist, to a patient in need thereof. In some embodiments, the method may reduce the frequency of kidney-related disease events. In some embodiments, the method may reduce the severity of kidney-related disease events. In some embodiments, the method described herein relates to delaying the clinical progression (worsening) of kidney disease. In some embodiments, the patient may further be administered one or more adjunctive therapies or active agents for treating kidney disease in addition to one or more activin and / or GDF antagonists. For example, the patient may also be administered one or more adjunctive therapies or active agents, such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, water pills or diuretics (optionally with a low-salt diet), statins, the hormone erythropoietin (optionally with iron supplements), intravenous (IV) fluid supplements, calcium and / or vitamin D supplements, phosphate binders, calcium, glucose or sodium polystyrene sulfonate (Kayexalate, Kionex), hemodialysis, peritoneal dialysis, and / or kidney transplants. Some exemplary pharmaceutical agents for kidney disease are Lasix® (furosemide), Demadex® (torsemide), Edecrine® (ethacrynic acid), and Edecrine sodium.In certain preferred embodiments, the activin and / or GDF antagonists to be used in accordance with the methods described herein are one or more of the following inhibitors (antagonists), or combinations of inhibitors (antagonists): activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF11, GDF3, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0005] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists, to be used according to the methods and uses described herein, is an agent that inhibits at least GDF11 (e.g., a GDF11 antagonist). The effect on GDF11 inhibition can be determined, for example, using a cell-based assay, such as those described herein (e.g., a Smad signaling reporter assay). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least GDF11. Ligand binding activity can be determined, for example, using a binding affinity assay, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, a variety of activin and / or GDF antagonists that inhibit GDF11 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibit GDF11 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0006] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent that inhibits at least GDF8 (e.g., a GDF8 antagonist). The effect on GDF8 inhibition can be determined, for example, using a cell-based assay, such as those described herein (e.g., a Smad signaling reporter assay). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least GDF8. Ligand binding activity can be determined, for example, using a binding affinity assay, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D As described herein, a variety of activin and / or GDF antagonists that inhibit GDF8 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, an activin and / or GDF antagonist, or combination of antagonists, that inhibits GDF8 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF11, GDF3, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0007] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent that inhibits at least GDF3 (e.g., a GDF3 antagonist). The effect on GDF3 inhibition can be determined, for example, using cell-based assays, such as those described herein (e.g., Smad signaling reporter assays). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least GDF3. Ligand binding activity can be determined, for example, using binding affinity assays, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, a variety of activin and / or GDF antagonists that inhibit GDF3 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibit GDF3 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0008] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent that inhibits at least GDF1 (e.g., a GDF1 antagonist). The effect on GDF1 inhibition can be determined, for example, using cell-based assays, such as those described herein (e.g., Smad signaling reporter assays). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least GDF1. Ligand binding activity can be determined, for example, using binding affinity assays, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D As described herein, various activin and / or GDF antagonists that inhibit BMP6 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibit GDF1 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0009] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent that inhibits at least Nodal (e.g., a Nodal antagonist). The effect on Nodal inhibition can be determined, for example, using cell-based assays, such as those described herein (e.g., Smad signaling reporter assays). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least Nodal. Ligand binding activity can be determined, for example, using binding affinity assays, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least Nodal. Ligand binding activity can be determined, for example, using binding affinity assays, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, a variety of activin and / or GDF antagonists that inhibit Nodal can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibit Nodal may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0010] In certain aspects, the activin and / or GDF antagonists or combination of antagonists to be used according to the methods and uses described herein are agents that inhibit at least cryptics (e.g., cryptic antagonists). The effect on cryptic inhibition can be determined, for example, using cell-based assays (e.g., Smad signaling reporter assays), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonists or combination of antagonists of the present disclosure can bind at least cryptics. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonists or combination of antagonists of the present disclosure can bind at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10-9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D As described herein, a variety of activin and / or GDF antagonists that inhibit cryptics can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibits cryptics may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic 1B, Smad2, and Smad3.

[0011] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent that inhibits at least Cryptic 1B (e.g., a Cryptic 1B antagonist). The effect on Cryptic 1B inhibition can be determined, for example, using cell-based assays, such as those described herein (e.g., Smad signaling reporter assays). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least Cryptic 1B. Ligand binding activity can be determined, for example, using binding affinity assays, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, a variety of activin and / or GDF antagonists that inhibit cryptic 1B can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, an activin and / or GDF antagonist, or combination of antagonists, that inhibits Cryptic 1B may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Smad2, and Smad3.

[0012] In certain aspects, the activin and / or GDF antagonists or combination of antagonists to be used according to the methods and uses described herein are agents (e.g., activin antagonists) that inhibit at least activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE). The effect on activin inhibition can be determined, for example, using cell-based assays (e.g., Smad signaling reporter assays), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonists or combination of antagonists of the present disclosure can bind to at least activin. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonists or combination of antagonists of the present disclosure can bind to at least activin. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, various activin and / or GDF antagonists that inhibit activin can be used according to the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptide, follistatin polypeptide, and FLRG polypeptide), antibodies, small molecules, nucleotide sequences, and combinations thereof.In certain embodiments, the activin and / or GDF antagonists that inhibit activin, or combinations of antagonists, can further inhibit one or more of the following: GDF8, GFD3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3. In certain preferred embodiments, the activin and / or GDF antagonist, or combination of antagonists, to be employed in accordance with the methods and uses described herein is an agent that inhibits at least activin B.

[0013] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent (e.g., an ActRII antagonist) that inhibits at least ActRII (e.g., ActRIIA and / or ActRIIB). The effect on ActRII inhibition can be determined, for example, using a cell-based assay (e.g., a Smad signaling reporter assay), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least ActRII. Ligand binding activity can be determined, for example, using a binding affinity assay, including those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10-7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D As described herein, various activin and / or GDF antagonists that inhibit ActRII can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibit ActRII may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0014] In certain aspects, the activin and / or GDF antagonist or combination of antagonists to be used according to the methods and uses described herein are agents that inhibit at least ALK4 (e.g., ALK4 antagonists). The effect on ALK4 inhibition can be determined, for example, using cell-based assays (e.g., Smad signaling reporter assays), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK4. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK4. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK4. -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, a variety of activin and / or GDF antagonists that inhibit ALK4 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, an activin and / or GDF antagonist, or combination of antagonists, that inhibits ALK4 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK5, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0015] In certain aspects, the activin and / or GDF antagonist or combination of antagonists to be used according to the methods and uses described herein are agents that inhibit at least ALK5 (e.g., ALK5 antagonists). The effect on ALK5 inhibition can be determined, for example, using cell-based assays (e.g., Smad signaling reporter assays), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK5. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK5. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D As described herein, a variety of activin and / or GDF antagonists that inhibit ALK5 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, the activin and / or GDF antagonist, or combination of antagonists, that inhibit ALK5 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK7, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0016] In certain aspects, the activin and / or GDF antagonist or combination of antagonists to be used according to the methods and uses described herein are agents that inhibit at least ALK7 (e.g., ALK7 antagonists). The effect on ALK7 inhibition can be determined, for example, using cell-based assays (e.g., Smad signaling reporter assays), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK7. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least ALK7. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, a variety of activin and / or GDF antagonists that inhibit ALK7 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, an activin and / or GDF antagonist, or combination of antagonists, that inhibits ALK7 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK5, ALK4, Cryptic, Cryptic 1B, Smad2, and Smad3.

[0017] In certain aspects, the activin and / or GDF antagonist, or combination of antagonists to be used according to the methods and uses described herein, is an agent that inhibits at least Smad2 (e.g., a Smad2 antagonist). The effect on Smad2 inhibition can be determined, for example, using a cell-based assay, such as those described herein (e.g., a Smad signaling reporter assay). Thus, in some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least Smad2. Ligand binding activity can be determined, for example, using a binding affinity assay, such as those described herein. In some embodiments, the activin and / or GDF antagonist, or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K D As described herein, a variety of activin and / or GDF antagonists that inhibit Smad2 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, an activin and / or GDF antagonist, or combination of antagonists, that inhibits Smad2 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, and Smad3.

[0018] In certain aspects, the activin and / or GDF antagonist or combination of antagonists to be used according to the methods and uses described herein are agents that inhibit at least Smad3 (e.g., Smad3 antagonists). The effect on Smad3 inhibition can be determined, for example, using cell-based assays (e.g., Smad signaling reporter assays), including those described herein. Thus, in some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least Smad3. Ligand binding activity can be determined, for example, using binding affinity assays, including those described herein. In some embodiments, the activin and / or GDF antagonist or combination of antagonists of the present disclosure can bind to at least 1×10 -7 M (e.g., at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, or at least 1 × 10 -12 M)K DAs described herein, various activin and / or GDF antagonists that inhibit Smad3 can be used in accordance with the methods and uses described herein, including, for example, ligand traps (e.g., ActRII polypeptides, follistatin polypeptides, and FLRG polypeptides), antibodies, small molecules, nucleotide sequences, and combinations thereof. In certain embodiments, an activin and / or GDF antagonist, or combination of antagonists, that inhibits Smad3 may further inhibit one or more of the following: activin (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF8, GDF3, GDF11, GDF1, Nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, Cryptic, Cryptic 1B, and Smad2.

[0019] In certain aspects, the activin and / or GDF antagonist that is the subject of use according to the methods and uses described herein is an ActRII polypeptide. The term "ActRII polypeptide" collectively refers to naturally occurring ActRIIA and ActRIIB polypeptides, as well as truncated forms and mutants thereof, such as those described herein. Preferably, the ActRII polypeptide comprises, consists essentially of, or consists of the ligand binding domain of the ActRII polypeptide or modified (mutant) forms thereof. For example, in some embodiments, the ActRIIA polypeptide comprises, consists essentially of, or consists of the ActRIIA ligand binding domain of the ActRIIA polypeptide, such as a portion of the ActRIIA extracellular domain. Similarly, the ActRIIB polypeptide comprises, consists essentially of, or consists of the ActRIIB ligand binding domain of the ActRIIB polypeptide, such as a portion of the ActRIIB extracellular domain. Preferably, the ActRII polypeptide that is the subject of use according to the methods described herein is a soluble polypeptide.

[0020] In certain aspects, the present disclosure relates to compositions comprising ActRIIA polypeptides and uses thereof. For example, in some embodiments, the ActRIIA polypeptides of the present disclosure comprise 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 the sequence of amino acids 30 to 110 of SEQ ID NO:9. In some embodiments, an ActRIIA polypeptide of the disclosure begins at a residue corresponding to any one of amino acids 21-30 of SEQ ID NO:9 (e.g., begins at any one of amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) and ends at a position corresponding to any one of amino acids 110-135 of SEQ ID NO:9 (e.g., amino acids 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 223, 224, 25, 26, 27, 28, 29, or 30). 8, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135), or 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. In other embodiments, an ActRIIA polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 9. In other embodiments, an ActRIIA polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 11. In yet other embodiments, the ActRIIA polypeptide may comprise 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 the amino acid sequence of SEQ ID NO:12.In still other embodiments, an ActRIIA polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 177. In still other embodiments, an ActRIIA polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 178. In yet other embodiments, the ActRIIA polypeptide may comprise, consist essentially of, or consist of 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 the amino acid sequence of SEQ ID NO:179.

[0021] In other aspects, the present disclosure relates to compositions comprising ActRIIB polypeptides and uses thereof. For example, in some embodiments, the ActRIIB polypeptide of the present disclosure comprises 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 the sequence of amino acids 29-109 of SEQ ID NO: 1. In some embodiments, the ActRIIB polypeptide may comprise 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 the sequence of amino acids 29-109 of SEQ ID NO: 1, where the ActRIIB polypeptide comprises an acidic amino acid [naturally occurring (E or D) or artificial acidic amino acid] at position 79 with respect to SEQ ID NO: 1. In other embodiments, the ActRIIB polypeptide comprises 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 the sequence of amino acids 25-131 of SEQ ID NO: 1. In some embodiments, the ActRIIB polypeptide may comprise 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 the sequence of amino acids 25-131 of SEQ ID NO: 1, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1.In some embodiments, an ActRIIB polypeptide may comprise 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 a sequence beginning with a residue corresponding to any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of SEQ ID NO:1 and ending with a residue corresponding to any one of amino acids 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:1. In other embodiments, the ActRIIB polypeptide begins at a residue corresponding to any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of SEQ ID NO:1 and includes any one of amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 of SEQ ID NO:1. 1, 130, 131, 132, 133, or 134, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1. In other embodiments, the ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 1. In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO:1, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 relative to SEQ ID NO:1.In yet other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 2. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 2, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In yet other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 3. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 3, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In yet other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 4. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 4, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.In yet other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 5. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 5, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In yet other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 6. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 6, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In yet other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 181. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 181, wherein the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 182. In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 182, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 184. In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 184, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1. In other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 187. In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 187, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 188. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 189. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 190. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 192. In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 192, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 193.In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 193, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 196. In some embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 196, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 197. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 198. In still other embodiments, an ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 199. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In yet other embodiments, the ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 201. In some embodiments, the ActRIIB polypeptide may comprise 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 the amino acid sequence of SEQ ID NO: 201, where the ActRIIB polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1.

[0022] As described herein, ActRII polypeptides and variants thereof can be homomultimers, such as homodimers, homotrimers, homotetramers, homopentamers, and higher order homomultimer complexes.In certain preferred embodiments, ActRII polypeptides and variants thereof are homodimers.In certain embodiments, the ActRII polypeptide dimers described herein comprise a first ActRII polypeptide covalently or non-covalently bound to a second ActRII polypeptide, where the first polypeptide comprises an ActRII domain and the amino acid sequence of a first member (or a second member) of an interaction pair (e.g., an immunoglobulin constant domain), and the second polypeptide comprises an ActRII polypeptide and the amino acid sequence of a second member (or a first member) of an interaction pair.

[0023] In certain aspects, the ActRII polypeptide, including its variants, can be a fusion protein. For example, in some embodiments, the ActRII polypeptide can be a fusion protein that includes an ActRII polypeptide domain and one or more heterologous (non-ActRII) polypeptide domains. In some embodiments, the ActRII polypeptide can be a fusion protein with an amino acid sequence from an ActRII polypeptide (e.g., the ligand-binding domain of an ActRII receptor or a variant thereof) as one domain, and one or more heterologous domains that provide desired properties, such as improved pharmacokinetics, easy purification, targeting to specific tissues, etc. For example, the domain of the fusion protein can enhance one or more of in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and / or purification. Optionally, the ActRII polypeptide domain of the fusion protein can be directly linked (fused) to one or more heterologous polypeptide domains, or an intervening sequence, such as a linker, can be located between the amino acid sequence of the ActRII polypeptide and the amino acid sequence of one or more heterologous domains. In certain embodiments, the ActRII fusion protein includes a relatively unstructured linker located between the heterologous domain and the ActRII domain. The unstructured linker can correspond to an approximately 15 amino acid unstructured region at the C-terminal end ("tail") of the extracellular domain of ActRII, or can be an artificial sequence of 3 to 15, 20, 30, 50 or more amino acids that is relatively free of secondary structure. The linker can be rich in glycine and / or proline residues and can include, for example, repeat sequences of threonine / serine and glycine. Examples of linkers include, but are not limited to, the sequences TGGG (SEQ ID NO: 217), GGG (SEQ ID NO: 223), GGGG (SEQ ID NO: 222), TGGGG (SEQ ID NO: 219), SGGGG (SEQ ID NO: 220), GGGGS (SEQ ID NO: 221), and SGGG (SEQ ID NO: 218).In some embodiments, the ActRII fusion protein can include an immunoglobulin constant domain, including, for example, the Fc portion of an immunoglobulin. For example, an amino acid sequence derived from the Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE, or IgM immunoglobulin. For example, the Fc portion of the immunoglobulin domain comprises, consists essentially of, or consists of 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 any one of SEQ ID NOs: 135-149. Such an immunoglobulin domain can include one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that affect altered Fc activity (e.g., one or more Fc effector functions). In some embodiments, the ActRII fusion protein comprises an amino acid sequence as represented by formula ABC. For example, the B portion is an N-terminally and C-terminally truncated ActRII polypeptide, for example, as described herein. The A and C portions can be independently 0, 1, or more amino acids, and. Both the A and C portions are heterologous to B. The A and / or C portions can be linked to the B portion via a linker sequence. In certain embodiments, the ActRII fusion protein comprises a leader sequence. The leader sequence can be a native ActRII leader sequence or a heterologous leader sequence. In certain embodiments, the leader sequence is a tissue plasminogen activator (TPA) leader sequence (e.g., SEQ ID NO: 215).

[0024] The ActRII polypeptide, including its variants, can include purification subsequences such as epitope tags, FLAG tags, polyhistidine sequences, and GST fusions. Optionally, the ActRII polypeptide includes one or more modified amino acid residues selected from the following modified amino acid residues: glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, and / or amino acids conjugated to lipid moieties. The ActRII polypeptide can have at least one N-linked sugar and can include two, three, or more N-linked sugars. Such polypeptides can also include O-linked sugars. In general, it is preferred that the ActRII polypeptide is expressed in a mammalian cell line that favorably mediates native glycosylation of the polypeptide to reduce the likelihood of an undesirable immune response in a patient. ActRII polypeptides can be produced in a variety of cell lines that glycosylate proteins in a manner suitable for patient use, including genetically engineered insect cells or yeast cells, as well as mammalian cells such as COS cells, CHO cells, HEK cells, and NSO cells. In some embodiments, the ActRII polypeptide is glycosylated and has a glycosylation pattern obtained from a Chinese Hamster Ovary cell line. In some embodiments, the ActRII polypeptides of the present disclosure exhibit a serum half-life in a mammal (e.g., mouse or human) of at least 4, 6, 12, 24, 36, 48, or 72 hours. Optionally, ActRII can exhibit a serum half-life in a mammal (e.g., mouse or human) of at least 6, 8, 10, 12, 14, 20, 25, or 30 days.

[0025] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising one or more ActRII antagonists of the present disclosure and pharma- ceutical acceptable carriers.The pharmaceutical compositions can also comprise one or more additional active agents, such as compounds used to treat kidney disease, particularly used to treat or prevent one or more complications of kidney disease (e.g., kidney tissue damage, fibrosis, and / or inflammation).Generally, the pharmaceutical compositions will preferably be pyrogen-free (meaning pyrogen-free to the extent required by the regulatory agency that controls the quality of products for therapeutic use).

[0026] In certain cases, when administering the ActRII antagonist or combination of antagonists of the present disclosure for the disorders or conditions described herein, it may be desirable to monitor the effect on red blood cells during the administration of the ActRII antagonist, or to determine or adjust the administration of the ActRII antagonist to reduce the undesirable effect on red blood cells.For example, the increase in red blood cell level, hemoglobin level, or hematocrit level may cause undesirable increase in blood pressure.

[0027] In certain aspects, the activin and / or GDF antagonist of interest used according to the disclosed method and use is an antibody, or a combination of antibodies.In some embodiments, the antibody binds to at least ActRII (ActRIIA and / or ActRIIB).In certain embodiments, the antibody that binds to ActRII inhibits ActRII signaling, optionally when measured in a cell-based assay such as that described herein.In certain embodiments, the antibody that binds to ActRII inhibits one or more GDF ligands, type I receptors, or co-receptors by binding to ActRII. In certain embodiments, the antibody that binds to ActRII inhibits one or more GDF ligands selected from the following from binding to ActRII: activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE), GDF8, GDF11, GDF1, nodal, GDF3, cryptic, cryptic 1B, ALK4, ALK5, ALK7, Smad2, and Smad3. In some embodiments, the antibody binds to at least ALK4. In certain embodiments, the antibody that binds to ALK4 inhibits ALK4 signal transduction, as measured in a cell-based assay, such as those described herein, optionally. In certain embodiments, the antibody that binds to ALK4 inhibits one or more GDF ligands, type II receptors, or co-receptors from binding to ALK4. In certain embodiments, the antibody that binds to ALK4 inhibits one or more GDF ligands selected from the following from binding to ALK4: activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE), GDF8, GDF11, GDF1, nodal, GDF3, ALK5, ALK7, cryptic, cryptic 1B, Smad2, and Smad3. In some embodiments, the antibody binds to at least ALK5.In certain embodiments, the antibody that binds to ALK5 inhibits ALK5 signal transduction, optionally when measured in a cell-based assay such as described herein. In certain embodiments, the antibody that binds to ALK5 inhibits one or more GDF ligands, type II receptors, or co-receptors from binding to ALK5. In certain embodiments, the antibody that binds to ALK5 inhibits one or more GDF ligands selected from the following from binding to ALK5: activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE), GDF8, GDF11, GDF1, nodal, GDF3, ALK4, ALK7, cryptic, cryptic 1B, Smad2, and Smad3. In some embodiments, the antibody binds to at least ALK7. In certain embodiments, the antibody that binds to ALK7 inhibits ALK7 signal transduction, optionally when measured in a cell-based assay such as described herein. In certain embodiments, the antibody that binds to ALK7 inhibits one or more GDF ligands, type II receptors, or co-receptors from binding to ALK7.In certain embodiments, the antibody that binds to ALK7 inhibits one or more GDF ligands selected from the following from binding to ALK7: activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE), GDF8, GDF11, GDF1, nodal, GDF3, ALK4, ALK5, cryptic, cryptic 1B, Smad2, and Smad3.In some embodiments, the antibody binds to at least GDF11.In certain embodiments, the antibody that binds to GDF11 inhibits ActRII signaling, as measured in a cell-based assay, such as that described herein, if desired. In certain embodiments, an antibody that binds GDF11 inhibits GDF11-ActRII binding and / or GDF11-ALK binding (eg, GDF11-ALK4, GDF11-ALK5, and / or GDF11-ALK7 binding).In some embodiments, the antibody binds at least GDF8. In certain embodiments, the antibody that binds GDF8 inhibits ActRII signaling, as measured in a cell-based assay, such as those described herein. In certain embodiments, the antibody that binds GDF8 inhibits GDF8-ActRII binding and / or GDF8-ALK binding (e.g., GDF8-ALK4, GDF8-ALK5, and / or GDF8-ALK7 binding). In some embodiments, the antibody binds at least GDF3. In certain embodiments, the antibody that binds GDF3 inhibits ActRII signaling, as measured in a cell-based assay, such as those described herein. In certain embodiments, the antibody that binds GDF3 inhibits GDF3-ActRII binding and / or GDF3-ALK binding (e.g., GDF3-ALK4, GDF3-ALK5, and / or GDF3-ALK7 binding). In some embodiments, the antibody binds to at least activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE). In certain embodiments, an antibody that binds to activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE) inhibits ActRII signaling, optionally as measured in a cell-based assay such as those described herein. In certain embodiments, an antibody that binds to activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE) inhibits activin-ActRII binding and / or activin-ALK binding (e.g., activin-ALK4, activin-ALK5, and / or activin-ALK7 binding). In some embodiments, the antibody binds to activin B. In certain embodiments, an antibody that binds to activin B inhibits ActRII signaling, optionally as measured in a cell-based assay such as those described herein.In certain embodiments, the antibody that binds to activin B inhibits activin B-ActRII binding and / or activin B-ALK binding (e.g., activin B-ALK4, activin B-ALK5, and / or activin B-ALK7 binding). In some embodiments, the antibody is a multispecific antibody or combination of multispecific antibodies that binds to one or more of ActRIIB, ActRIIA, ALK4, ALK5, ALK7, GDF11, GDF8, activin, GDF1, nodal, GDF3, cryptic, cryptic 1B, Smad2, and Smad3. In certain aspects, the multispecific antibody or combination of multispecific antibodies inhibits signal transduction in one or more of the following cell-based assays: ActRIIB, GDF11, GDF8, activin, GDF3, nodal, GDF1, cryptic, cryptic 1B, Smad2, and Smad3. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is a single chain antibody, F(ab'). 2 The antibody is preferably a fusion protein comprising a single chain diabody, a tandem single chain Fv fragment, a tandem single chain diabody, or a fusion protein comprising a single chain diabody and at least a portion of an immunoglobulin heavy chain constant region.

[0028] In certain aspects, the activin and / or GDF antagonist is a small molecule inhibitor or a combination of small molecule inhibitors. In some embodiments, the small molecule inhibitor is an inhibitor of at least ActRII (e.g., ActRIIA and / or ActRIIB). In some embodiments, the small molecule inhibitor is an inhibitor of at least ALK4. In some embodiments, the small molecule inhibitor is an inhibitor of at least ALK5. In some embodiments, the small molecule inhibitor is an inhibitor of at least ALK7. In some embodiments, the small molecule inhibitor is an inhibitor of at least GDF11. In some embodiments, the small molecule inhibitor is an inhibitor of at least GDF8. In some embodiments, the small molecule inhibitor is an inhibitor of at least GDF1. In some embodiments, the small molecule inhibitor is an inhibitor of at least Nodal. In some embodiments, the small molecule inhibitor is an inhibitor of at least Cryptic. In some embodiments, the small molecule inhibitor is an inhibitor of at least Cryptic 1B. In some embodiments, the small molecule inhibitor is an inhibitor of at least Smad2. In some embodiments, the small molecule inhibitor is an inhibitor of at least Smad3. In some embodiments, the small molecule inhibitor is at least an inhibitor of GDF3.In some embodiments, the small molecule inhibitor is at least an inhibitor of activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE and activin BE).In some embodiments, the small molecule inhibitor is at least an inhibitor of activin B.

[0029] In certain aspects, the activin and / or GDF antagonist is a nucleic acid inhibitor or a combination of nucleic acid inhibitors. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least ActRII (e.g., ActRIIA and / or ActRIIB). In some embodiments, the nucleic acid inhibitor is an inhibitor of at least ALK4. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least ALK5. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least ALK7. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF11. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF8. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF1. In some embodiments, the small molecule inhibitor is an inhibitor of at least nodal. In some embodiments, the small molecule inhibitor is an inhibitor of at least cryptic. In some embodiments, the small molecule inhibitor is an inhibitor of at least cryptic 1B. In some embodiments, the small molecule inhibitor is an inhibitor of at least Smad2. In some embodiments, the small molecule inhibitor is an inhibitor of at least Smad3. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least GDF3. In some embodiments, the nucleic acid inhibitor is an inhibitor of at least activin (e.g., activin A, activin B, activin C, activin AB, activin AC, activin BC, activin E, activin AE, and activin BE). In some embodiments, the nucleic acid inhibitor is an inhibitor of at least activin B.

[0030] In certain aspects, the activin and / or GDF antagonist is a follistatin polypeptide. In some embodiments, the follistatin polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 150. In some embodiments, the follistatin polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 151. In some embodiments, the follistatin polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 152. In some embodiments, the follistatin polypeptide comprises 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 the amino acid sequence of SEQ ID NO: 153. In some embodiments, the follistatin polypeptide comprises 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 at least one of the amino acid sequences set forth in SEQ ID NOs: 154-160.

[0031] In certain aspects, the activin and / or GDF antagonist is a FLRG polypeptide. In some embodiments, the FLRG polypeptide comprises 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 at least one of the amino acid sequences of SEQ ID NOs: 161-164.

[0032] In certain aspects, the activin and / or GDF antagonist is a WFIKKN1 polypeptide. In some embodiments, the WFIKKN1 polypeptide comprises 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 at least one of the amino acid sequences of SEQ ID NOs: 165-167.

[0033] In certain aspects, the activin and / or GDF antagonist is a WFIKKN2 polypeptide. In some embodiments, the WFIKKN1 polypeptide comprises 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 at least one of the amino acid sequences of SEQ ID NOs: 168-172.

[0034] In certain aspects, the activin and / or GDF antagonist is a lefty polypeptide. In some embodiments, the WFIKKN1 polypeptide comprises 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 at least one of the amino acid sequences of SEQ ID NOs: 173-174.

[0035] In certain aspects, the activin and / or GDF antagonist is a Cerbrus polypeptide. In some embodiments, the WFIKKN1 polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence to at least one of the amino acid sequences of SEQ ID NO: 175.

[0036] In certain aspects, the activin and / or GDF antagonist is a Coco polypeptide. In some embodiments, the WFIKKN1 polypeptide comprises 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 at least one of the amino acid sequences of SEQ ID NO: 176.

[0037] In certain embodiments, the present disclosure relates to the use of one or more activin and / or GDF antagonists in the manufacture of a medicament for treating, preventing, or reducing the progression rate and / or severity of one or more complications of kidney disease or kidney disease described herein, optionally in combination with one or more other adjunctive therapies or active agents for treating kidney disease.In certain embodiments, the present disclosure relates to the use of one or more activin and / or GDF antagonists in the manufacture of a medicament for treating, preventing, or reducing the progression rate and / or severity of one or more complications of kidney disease or kidney disease described herein, optionally in combination with one or more other adjunctive therapies or active agents for treating kidney disease.

[0038] The present disclosure provides at least combinations of agents (e.g., cell signaling antagonists) for therapeutic use. Such antagonists include at least one of activin and / or growth and differentiation factor (GDF) antagonists, including, for example, activin, GDF8, GDF11, GDF3, GDF1, Nodal, activin receptor type IIA (ActRIIA), ActRIIB, ALK4, ALK5, ALK7, Cripto-1, cryptic, cryptic 1B, Smad2, and Smad3. The agents can form multimeric complexes with each other through at least one covalent or non-covalent bond. Exemplary structures of some of these multimeric complexes are shown in Figures 5 and 6. An exemplary list of possible dimers of such agents is given below:

[0039] Possible heterodimers : I. Type I-II heterodimer: ALK4:ActRIIB;ALK4:ActRIIA;ALK4:BMPRII;ALK4:MISRII;ALK5:ActRIIB;ALK5:ActRIIA;ALK5: BMPRII;ALK5:MISRII;ALK7:ActRIIB;ALK7:ActRIIA;ALK7:BMPRII;ALK7:MISRII;ALK1:ActRIIB;ALK1:ActRIIA

[0040] II. Type I - Type I heterodimer: ALK1:ALK4; ALK1:ALK5; ALK1:ALK7; ALK4:ALK5; ALK4:ALK7; ALK5:ALK7

[0041] III. Type II-Type II heterodimer: ActRIIA:ActRIIB;ActRIIA:BMPRII;ActRIIA:MISRII;ActRIIB:BMPRII;ActRIIB:MISRII

[0042] IV. Coreceptor heterodimers: cryptic:crypt;cryptic:cryptic 1B;crypt:cryptic 1B;ALK1:cryptic;ALK1:cryptic 1B;ALK1:cryptic;ALK4:cryptic;ALK4:cryptic 1B;ALK4:cryptic;ALK5:cryptic;ALK5:cryptic 1B;ALK5:cryptic;ALK7:cryptic;ALK7:cryptic 1B;ALK7:crypt;ActRIIA:cryptic;ActRIIA:cryptic 1B;ActRIIA:cryptic;ActRIIB:cryptic;ActRIIB:cryptic 1B;ActRIIB:crypto;BMPRII:cryptic;BMPRII:cryptic1B;BMPRII:crypto;MISRII:cryptic;MISRII:cryptic1B;MISRII:crypto

[0043] Possible homodimers : ALK4:ALK4;ALK5:ALK5;ALK7:ALK7;ActRIIA:ActRIIA;ActRIIB:ActRIIB;Crypto:Crypto;Cryptic1B:Cryptic1B;Cryptic-Cryptic

[0044] Other antagonists as monodimers for dimerization : Inhibitors (e.g., antibodies, small molecules, RNA interference, etc.) of ligands (e.g., activin A, B, C, and E; GDF8; GDF11; GDF3; GDF1; and nodal) Inhibitors of type I receptors (e.g., ALK4, ALK5, and ALK7) (e.g., antibodies, small molecules, RNA interference, etc.) Inhibitors of type II receptors (e.g., ActRIIA and ActRIIB) (e.g., antibodies, small molecules, RNA interference, etc.)

[0045] Inhibitors (e.g., antibodies, small molecules, RNA interference, etc.) of co-receptors (e.g., Crypto, Cryptic, and Cryptic1B) Inhibitors of Smad proteins (e.g., Smad2 and Smad3) (e.g., antibodies, small molecules, RNA interference, etc.) Natural ligand traps (naturally occurring proteins that bind to one or more activin / GDF proteins) (e.g., WFIKKN1, WFIKKN2, FST, FLRG, Dan-related proteins Cerberus and Coco, Lefty A, Lefty B, WFIKKN1 and WFIKKN2).

[0046] Oligomers and multimers can be formed using at least the same strategy as shown in Figures 5 and 6 herein. For example, tetramers can be formed using two identical or different heterodimers or homodimers. The monomers can be the listed antagonists themselves, or can include fusion proteins that include the listed antagonists. For example, the monomers can include fusion proteins of ALK4 and Fc domains to produce homodimers of ALK4-Fc:ALK4-Fc, or heterodimers of ALK4-Fc and other agents (e.g., ActBRIIA-Fc). In addition, ALK4 can be fused to itself or other agents (e.g., ALK5) as a monomer to produce homodimers of ALK4-ALK4:ALK4-ALK4 or ALK4-ALK5:ALK4-ALK5, or heterodimers that include ALK4-ALK4 or ALK4-ALK5. Thus, possible dimers also include those containing the same agent fused in different orientations, for example, ALK4-ALK5:ActRIIA-ActRIIB and ALK5-ALK4:ActRIIA:ActRIIB can form different heterodimer structures. [Brief description of the drawings]

[0047] [Figure 1] This shows an alignment of the extracellular domains of human ActRIIA (sequence number 11) and human ActRIIB (sequence number 3), with residues for direct contact with the ligand shown in boxes, as predicted herein based on a combined analysis of multiple ActRIIB and ActRIIA crystal structures. [Diagram 2] This figure shows a multiple sequence alignment of various vertebrate ActRIIB precursor proteins that do not contain its intracellular domain (including human ActRIIB precursor protein that does not contain its intracellular domain (sequence number 16)), human ActRIIA precursor protein that does not contain its intracellular domain (sequence number 15), and consensus ActRII precursor protein that does not contain the intracellular domain (sequence number 17). [Diagram 3]FIG. 1 shows a multiple sequence alignment of the extracellular domain of various vertebrate ActRIIA proteins and human ActRIIA (sequence number 11). [Figure 4] Figure 1 shows a multiple sequence alignment of Fc domains from human IgG isotypes using Clustal 2.1. The hinge region is indicated by a dotted underline. Double underlines indicate examples of positions engineered in IgG1 Fc to promote asymmetric chain pairing, and corresponding positions for the other isotypes IgG2, IgG3 and IgG4. [Figure 5-1]AD: Schematic example of a heteromeric protein complex comprising a type I receptor polypeptide (denoted as "I") (e.g., a polypeptide that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK1, ALK4, ALK5, or ALK7 protein from human or other species such as those described herein) and a type II receptor polypeptide (denoted as "II") (e.g., a polypeptide that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIA, ActRIIB, MISRII, or BMPRII protein from human or other species such as those described herein). In the illustrated embodiment, 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 the interaction pair ("C2"). Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A): 95-106). In each fusion polypeptide, a linker can be located between the type I receptor polypeptide or the type II receptor polypeptide and the corresponding member of the interaction pair. The first and second members of the interaction pair are unguided, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and can have the same or different amino acid sequences. See FIG. 5A. Alternatively, the interaction pair can be a induced (asymmetric) pair, meaning that the members of the pair associate with each other preferentially over self-association. See FIG. 5B. Higher order complexes can also be envisioned. See FIGS. 5C and 5D. [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 6-1]AG: A schematic example of a heteromeric protein complex comprising two type I receptor polypeptides (denoted as "I") (e.g., polypeptides that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK1, ALK4, ALK5, or ALK7 protein from a human or other biological species such as those described herein) and two type II receptor polypeptides (denoted as "II") (e.g., polypeptides that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIA, ActRIIB, MISRII, or BMPRII protein from a human or other biological species such as those described herein). In exemplary embodiment 6A, the first type I receptor polypeptide (from left to right) is part of a fusion polypeptide that includes a first member of an interaction pair ("C1") and further includes an additional first member of the interaction pair ("A1"); the second type I receptor polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("C2") and further includes an additional first member of the interaction pair ("A2"). The first type II receptor polypeptide (from left to right) is part of a fusion polypeptide that includes a second member of the interaction pair ("B1"); the second type II receptor polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("B2"). A1 and A2 can be the same or different; B1 and B2 can be the same or different; C1 and C2 can be the same or different. In each fusion polypeptide, linkers can be located between the type I receptor polypeptide or the type II receptor polypeptide and the corresponding members of the interaction pair, as well as between the interaction pairs. FIG. 6A is an example of an association of an uninduced interaction pair, which means that the members of the pair can associate with each other or self-associate without substantial preference and can have the same or different amino acid sequences.In exemplary embodiment 6B, the first type II receptor polypeptide (from left to right) is part of a fusion polypeptide that includes a first member of an interaction pair ("C1") and further includes an additional first member of the interaction pair ("A1"); the second type II receptor ActRIIB polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("B2"). The first type I receptor polypeptide (from left to right) is part of a fusion polypeptide that includes a second member of the interaction pair ("B1"); the second type I receptor polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("C2") and further includes a first member of the interaction pair ("A2"). In each fusion polypeptide, a linker can be located between the type I receptor or type II receptor polypeptide and the corresponding member of the interaction pair, as well as between the interaction pairs themselves. Figure 6B is an example of an induced (asymmetric) interaction pair association, which means that the members of the pair preferentially associate with each other over self-association. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A): 95-106). Higher order complexes can also be envisioned. See Figures 6C-6F. Using similar methods, particularly those utilizing light and / or heavy chain immunoglobulins, truncations, or variants thereof, interaction pairs can be used to generate heterodimers similar to antibody Fab and F(ab')2 complexes (e.g., Spiess et al. (2015) Molecular Immunology 67(2A): 95-106). See Figure 6G. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 6-5] Continued from Figure 6-4. [Figure 6-6] Continued from Figure 6-5. [Figure 7] FIG. 7A shows purification of ActRIIA-hFc expressed in CHO cells visualized by sizing column (FIG. 7A) and Coomassie-stained SDS-PAGE (FIG. 7B, left lane: molecular weight standards; right lane: ActRIIA-hFc). [Figure 8] FIG. 8 shows the binding of ActRIIA-hFc to activin (FIG. 8A) and GDF-11 (FIG. 8B) as measured by Biacore™ assay. [Figure 9]FIG. 1 shows a schematic example of a heteromeric protein complex comprising an ALK4 polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ALK4 protein from a human or other species such as those described herein), an ActRIIB polypeptide (e.g., a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identical to the extracellular domain of an ActRIIB protein from a human or other species such as those described herein), and a ligand-binding domain of an antibody (e.g., a ligand-binding domain from an antibody that binds to one or more ALK4:ActRIIB-binding ligands). In an exemplary embodiment, the ALK4 polypeptide is part of a fusion polypeptide that includes a first member of an interaction pair ("C1") and further includes an additional first member of the interaction pair ("A1"). The ActRIIB polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("B1"). The heavy chain variable (VH) polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("C2") and further includes an additional first member of the interaction pair ("A2"). The heavy chain variable (VL) polypeptide is part of a fusion polypeptide that includes a second member of the interaction pair ("B2"). In each fusion polypeptide, linkers can be located between the ALK4 or ActRIIB polypeptide and the corresponding members of the interaction pair, between the interaction pairs, and between the VH and VL polypeptides and the members of the interaction pair. A1 and A2 can be the same or different; B1 and B2 can be the same or different; C1 and C2 can be the same or different.Suitable interaction pairs include, for example, constant heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A): 95-106). Figure 9A is an example of an induced (asymmetric) interaction pair association, meaning that the members of the pair associate with each other preferentially over self-association. Figure 9B is an example of an uninduced interaction pair association, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and can have the same or different amino acid sequences. Such antibody-ALK4:ActRIIB complexes may be useful in situations where it is desirable to further bind / antagonize agents that are not ALK4:ActRIIB ligands. Alternatively, such antibody-ALK4:ActRIIB complexes may be useful in situations where it is desirable to further enhance ALK4:ActRIIB ligand binding / antagonism. For example, as demonstrated by the examples herein, activin B, activin A, GDF11 and GDF8 all bind to ALK4:ActRIIB heterodimer with strong affinity.In addition, BMP6 binds to ALK4:ActRIIB heterodimer, but with relatively weak affinity.In certain situations where it is desirable to antagonize BMP6 activity, in addition to one or more of high affinity binding ligands (e.g., activin B, activin A, GDF11 and GDF8), BMP6 can compete for binding to ALK4:ActRIIB heterodimer.In such situations, the addition of the BMP6 binding domain of an antibody to ALK4:ActRIIB heteromultimer complex will improve the ability of such protein complex to antagonize BMP6 in addition to one or more of activin B, activin A, GDF11 and GDF8. [Figure 10]1 shows a schematic example of ALK4:ActRIIB single trap polypeptide. ALK4:ActRIIB single trap polypeptide can contain multiple ALK4 domains (for example, 1, 2, 3, 4, 5, 6, 7, 9, 10 or more domains) with the same or different sequences, and multiple ActRIIB domains (for example, 1, 2, 3, 4, 5, 6, 7, 9, 10 or more domains) with the same or different sequences. These ALK4 domains and ActRIIB domains can be arranged in any order, and can include one or more linker domains located between one or more of the ALK4 domains and ActRIIB domains. Such ligand traps can be used as therapeutic agents for treating or preventing diseases or conditions described herein. [Figure 11-1]AD: Schematic example of a multimeric protein complex comprising at least one ALK4:ActRIIB single-chain trap polypeptide. In exemplary embodiments 11A and 11B, the first ALK4:ActRIIB single-chain trap polypeptide (from left to right) is part of a fusion polypeptide comprising a first member ("C1") of an interaction pair, and the second ALK4:ActRIIB single-chain trap polypeptide is part of a fusion polypeptide comprising a second member ("C2") of an interaction pair. C1 and C2 can be the same or different. The first and second ALK4:ActRIIB single-chain trap polypeptides can be the same or different. In each fusion polypeptide, a linker can be located between the ALK4:ActRIIB single-chain trap polypeptide and the corresponding member of the interaction pair. Suitable interaction pairs include, for example, heavy and / or light chain immunoglobulin interaction pairs, truncations, and variants thereof, such as those described herein (e.g., Spiess et al. (2015) Molecular Immunology 67(2A): 95-106). Figure 11A is an example of an association of an uninduced interaction pair, meaning that the members of the pair can associate with each other or self-associate without substantial preference, and can have the same or different amino acid sequences. Figure 11B is an example of an association of an induced (asymmetric) interaction pair, meaning that the members of the pair associate with each other preferentially over self-association. Higher order complexes can also be envisioned. In addition, such ALK4:ActRIIB single-chain trap polypeptides can similarly associate, covalently or non-covalently, with one or more ALK4 polypeptides and / or one or more ActRIIB polypeptides. See Figure 11C. Such ALK4:ActRIIB single-chain trap polypeptides can also be covalently or non-covalently associated with the ligand-binding domain of one or more antibodies (e.g., the ligand-binding domain of an antibody that binds to one or more ALK4:ActRIIB binding ligands). See Figure 11D. [Figure 11-2]Continued from Figure 11-1. [Figure 12] Figure 1 shows comparative ligand binding data for ALK4-Fc:ActRIIB-Fc heterodimer protein complexes compared to ActRIIB-Fc homodimers and ALK4-Fc homodimers. For each protein complex, ligands are ranked by koff, the rate constant that correlates well with ligand signaling inhibition, and listed in descending order of binding affinity (the tightest binding ligands are listed at the top). On the left, yellow, red, green, and blue lines indicate the magnitude of the off-rate constants. Black solid lines indicate ligands that have strong or unchanged binding to the heterodimer compared to the homodimer, and red dashed lines indicate substantially reduced binding compared to the homodimer. As shown, the ALK4-Fc:ActRIIB-Fc heterodimer exhibits enhanced binding to activin B compared to either homodimer, retains strong binding to activin A, GDF8, and GDF11 as observed with the ActRIIB-Fc homodimer, and exhibits substantially reduced binding to BMP9, BMP10, and GDF3. Similar to the ActRIIB-Fc homodimer, the heterodimer retains a moderate level of binding to BMP6. [Figure 13]Figure 4 shows the IC50 comparison data of ALK4-Fc:ActRIIB-Fc heterodimer / ActRIIB-Fc:ActRIIB-Fc homodimer as determined by A-204 reporter gene assay as described herein.ALK4-Fc:ActRIIB-Fc heterodimer inhibits activin A, activin B, GDF8 and GDF11 signaling pathways similar to ActRIIB-Fc:ActRIIB-Fc homodimer.However, the inhibition of BMP9 and BMP10 signaling pathways by ALK4-Fc:ActRIIB-Fc heterodimer is significantly reduced compared to ActRIIB-Fc:ActRIIB-Fc homodimer. These data demonstrate that the ALK4:ActRIIB heterodimer is a more selective antagonist of activin A, activin B, GDF8, and GDF11 compared to the corresponding ActRIIB:ActRIIB homodimers. [Figure 14-1] AC: Gene expression profiles of fibrotic genes (Col1a1, fibronectin, PAI-1, CTGF, and a-SMA), inflammatory genes (TNF-α, and MCP1), cytokine genes (TGF-β1, TGF-β2, TGF-β3, and activin A), nephrotoxicity genes (NGAL), hypoxia-inducible factor 1-α (HIF1a), and activin A receptor (Acvr2A) from mouse kidneys subjected to unilateral ureteral obstruction (UUO). Samples from the contralateral non-operated kidney were used as control (Ctrl). Gene expression profiles were acquired 17 days after surgery. Mice received PBS or ALK4-Fc:ActRIIB-Fc homodimer on days 3, 7, 10, and 14 after surgery. ($) represents the statistical difference between UUO kidneys at 17 days in mice receiving PBS alone compared to UUO kidneys at 17 days in mice receiving ALK7-Fc:ActRIIB-Fc homodimer. (@) represents no transcript detected. [Figure 14-2] Continued from Figure 14-1. [Figure 15]Figure 1 shows comparative ligand binding data for ALK7-Fc:ActRIIB-Fc heterodimer protein complexes compared to ActRIIB-Fc homodimers and ALK7-Fc homodimers. For each protein complex, ligands are ranked by koff, the rate constant that correlates well with ligand signaling inhibition, and listed in descending order of binding affinity (tightest binding ligands are listed at the top). On the left, yellow, red, green, and blue lines indicate the magnitude of the off-rate constants. Black solid lines indicate ligands that have strong or unchanged binding to the heterodimer compared to the homodimer, and red dashed lines indicate substantially reduced binding compared to the homodimer. As shown, four of the five ligands with strong binding to the ActRIIB-Fc homodimer (activin A, BMP10, GDF8, and GDF11) show reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer, with the exception of activin B, which retains tight binding to the heterodimer. Similarly, three of the four ligands with moderate binding to the ActRIIB-Fc homodimer (GDF3, BMP6, and especially BMP9) show reduced binding to the ActRIIB-Fc:ALK7-Fc heterodimer and increased binding to activin AC, making it the second strongest ligand interaction with the heterodimer overall. Finally, activin C and BMP5 unexpectedly bind with moderate strength to the ActRIIB-Fc:ALK7 heterodimer, but have no (activin C) or weak (BMP5) binding to the ActRIIB-Fc homodimer. None of the ligands tested bind to the ALK7-Fc homodimer. [Figure 16A]Figure 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, fibronectin, PAI-1, CTGF, and a-SMA), inflammatory genes (TNF-α, and MCP1), cytokine genes (Tgfb1, Tgfb2, Tgfb3, and activin A), nephrotoxicity genes (NGAL), hypoxia-inducible factor 1-α (HIF1a), and activin A receptor (Acvr2A) from mouse kidneys subjected to unilateral ureteral obstruction (UUO). Samples from the contralateral non-operated kidney were used as control (Ctrl). Gene expression profiles were obtained 3 and 17 days after surgery. Mice received PBS or ALK7-Fc:ActRIIB-Fc homodimer on days 3, 7, 10, and 14 after surgery. Statistical analysis was performed using one-way ANOVA followed by Tukey analysis. (*) represents statistical difference between (i) control samples compared to UUO kidneys at 3 days or (ii) control samples compared to UUO kidneys at 17 days in mice administered ALK7-Fc:ActRIIB-Fc homodimer. ($) represents statistical difference between UUO kidneys at 17 days in mice administered PBS only compared to UUO kidneys at 17 days in mice administered ALK7-Fc:ActRIIB-Fc homodimer. (@) represents transcript not detected. [Figure 16B] Continued from Figure 16A. [Figure 16C] Continued from Figure 16B. [Figure 17] Figure 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIa-Fc homodimer. [Figure 18] FIG. 1 shows gene expression profiles of inflammatory genes (IL-1B and TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIa-Fc homodimer. [Figure 19]FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 and activin A) and renal injury genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIa-Fc homodimer. [Figure 20] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIB(20-134)-Fc homodimer. [Figure 21] FIG. 1 shows gene expression profiles of inflammatory genes (IL-1B and TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIB(20-134)-Fc homodimer. [Figure 22] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIB(20-134)-Fc homodimer. [Diagram 23] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-TGF-β1 / 2 / 3 pan-antibody (i.e., binds to TGF-β isoforms 1, 2, and 3). [Figure 24] FIG. 1 shows gene expression profiles of inflammatory genes (TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with anti-TGF-β1 / 2 / 3 pan-antibody (i.e., binds to TGF-β isoforms 1, 2 and 3). [Diagram 25] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-TGF-β1 / 2 / 3 pan-antibody (i.e., binds TGF-β isoforms 1, 2 and 3). [Figure 26] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-activin A antibody. [Figure 27] FIG. 1 shows gene expression profiles of inflammatory genes (IL-1B and TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with anti-activin A antibody. [Figure 28] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-activin A antibody. [Figure 29] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-activin A / B antibody. [Diagram 30] FIG. 1 shows gene expression profiles of inflammatory genes (IL-1B and TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with anti-activin A / B antibody. [Diagram 31] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-activin A / B antibody. [Diagram 32] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-activin B antibody. [Diagram 33] FIG. 1 shows gene expression profiles of inflammatory genes (IL-1B and TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with anti-activin B antibody. [Diagram 34]FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-activin B antibody. [Diagram 35] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIA antibody. [Diagram 36] FIG. 1 shows gene expression profiles of inflammatory genes (TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with anti-ActRIIA antibody. [Figure 37] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIA antibody. [Figure 38] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIA / IIB antibody. [Figure 39] FIG. 1 shows gene expression profiles of inflammatory genes (TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with anti-ActRIIA / IIB antibody. [Diagram 40] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIA / IIB antibody. [Diagram 41] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIB antibody. [Diagram 42] FIG. 1 shows gene expression profiles of inflammatory genes (TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIB antibody. [Diagram 43] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with anti-ActRIIB antibody. [Diagram 44] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIB(L79D, 25-131)-hFc homodimer. [Diagram 45] FIG. 1 shows gene expression profiles of inflammatory genes (IL-1B and TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIB(L79D, 25-131)-hFc homodimer. [Diagram 46] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ActRIIB(L79D, 25-131)-hFc homodimer. [Figure 49]Figure 1 shows the ligand binding data for ActRIIA-Fc:ALK4-Fc heterodimer protein complexes compared to ActRIIA-Fc homodimer and ALK4-Fc homodimer. As shown, ActRIIA-Fc:ALK4-Fc heterodimer shows enhanced binding to activin A, especially to activin AC, and retains strong binding to activin AB and GDF11, compared to ActRIIA-Fc homodimer. In addition, activin B, the ligand with the highest affinity for ActRIIA-Fc homodimer, shows reduced affinity (but still within the high affinity range) to ActRIIA-Fc:ALK4-Fc heterodimer. ActRIIA-Fc:ALK4-Fc heterodimer also shows significantly reduced binding to BMP10 compared to ActRIIA-Fc homodimer. [Figure 50] FIG. 1 shows gene expression profiles of fibrotic genes (Col1a1, Col3a1, PAI-1, fibronectin, CTGF, and a-SMA) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ALK4-Fc:ActRIIA-Fc heterodimer. [Figure 51] FIG. 1 shows gene expression profiles of inflammatory genes (TNF-α) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) following treatment with ALK4-Fc:ActRIIA-Fc heterodimer. [Figure 52] FIG. 1 shows gene expression profiles of cytokine genes (Tgfb1 / 2 / 3 and activin A) and nephrotoxicity genes (NGAL) from mouse kidneys subjected to unilateral ureteral obstruction (UUO) after treatment with ALK4-Fc:ActRIIA-Fc heterodimer. [Figure 53]Figure 1 shows the ligand binding data for the BMPRII-Fc:ALK4-Fc heterodimer protein complex compared to BMPRII-Fc homodimer and ALK4-Fc homodimer.BMPRII-Fc:ALK4-Fc heterodimer differs from both homodimers in that it binds to several activin ligands with high or moderate strength, and differs from BMPRII-Fc homodimer in that it only binds weakly to BMP15.Most notably, BMPRII-Fc:ALK4-Fc heterodimer binds to the heterodimer ligand activin AB strongly and with high selectivity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] 1. Overview The TGF-β superfamily includes more than 30 secreted factors, including TGF-β, activin, nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and anti-Mullerian hormone (AMH) (Weiss et al. (2013) Developmental Biology, 2(1): 47-63). Found in both vertebrates and invertebrates, members of this superfamily are ubiquitously expressed in a wide range of tissues and function in the earliest stages of development throughout the life of an animal. Indeed, TGF-β superfamily proteins are key mediators of stem cell self-renewal, gastrulation, differentiation, organ morphogenesis, and adult tissue homeostasis. Consistent with this ubiquitous activity, aberrant TGF-β superfamily signaling is associated with a wide range of human pathologies.

[0049] Ligands of the TGF-β superfamily share the same dimeric structure in which the central 3-1 / 2 helix of one monomer packs into a concave surface formed by the β-strands of the other monomer. The majority of TGF-β family members are further stabilized by an intermolecular disulfide bond that traverses through a ring formed by two additional 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).

[0050] TGF-β superfamily signaling is mediated by heteromeric complexes of type I and type II serine / threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins 1, 2, 3, 5, and 8) upon ligand stimulation [Massague (2000) Nat. Rev. Mol. Cell Biol. 1:169-178]. These type I and type II receptors are transmembrane proteins composed of a ligand-binding extracellular domain containing a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase specificity. In general, type I receptors mediate intracellular signaling, while type II receptors are required for binding of TGF-β superfamily ligands. Type I and type II receptors form a stable complex after ligand binding, which results in phosphorylation of type I receptors by type II receptors.

[0051] The TGF-β family can be divided into two phylogenetic branches based on the type I receptors they bind and the Smad proteins they activate. One branch is a relatively recently evolved branch, including, for example, TGF-β, activin, GDF8, GDF9, GDF11, BMP3, and nodal, which signal through type I receptors to activate Smad2 and Smad3 (Hinck (2012) FEBS Letters 586:1860-1870). The other branch includes more distantly related proteins of the superfamily, including, for example, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7, which signal through Smad1, Smad5, and Smad8.

[0052] Activins are members of the TGF-β superfamily, originally discovered as regulators of follicle-stimulating hormone secretion, but have since been characterized for a variety of reproductive and non-reproductive roles. There are three basic activin forms (A, B, and AB), which consist of two closely related β subunits (β A β A , β B β B , and β A β B Activin C and activin E are also encoded in the human genome, and are expressed primarily in the liver and are involved in the regulation of β C or β EHeterodimeric forms containing activin are also known. Within the TGF-β superfamily, activin is a unique and multifunctional factor that stimulates hormone production in ovarian and placental cells, supports neural cell survival, positively or negatively affects cell cycle progression depending on the cell type, and can induce mesoderm 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 gland, activin promotes FSH synthesis and secretion, whereas inhibin reduces FSH synthesis and secretion. Other proteins that regulate activin bioactivity and / or bind to activin include follistatin (FS), follistatin-related protein (FSRP, also known as FLRG or FSTL3), and α 2 - Macroglobulins are included.

[0053] As described herein, agents that bind to "activin A" include isolated β A In the context of subunits or dimeric complexes (e.g., β A β A Homodimer or β A β B Heterodimers) or as A Agents that specifically bind to the subunits of heterodimeric complexes (e.g., β A β B In the case of activin A, the drug that binds to β A Specific for epitopes present within the subunits, but not the non-β subunits of the complex A Subunits (e.g., β of the complex) BSimilarly, the agents disclosed herein that antagonize "activin A" do not bind to epitopes present within isolated β A In the context of subunits or dimeric complexes (e.g., β A β A Homodimer or β A β B Heterodimers) or as A These are agents that inhibit one or more activities mediated by the β subunit. A β B In the case of the heterodimer, drugs that inhibit "activin A" inhibit β A Specifically inhibit the activity of one or more of the subunits, but not the non-β subunits of the complex. A Subunits (e.g., β of the complex) B Subunits) that do not inhibit the activity of activin B, activin C, and activin E. The agents disclosed herein that antagonize activin AB are agents that bind to and / or inhibit the activity of β A One or more activities mediated by the subunits and B An agent that inhibits one or more of the activities mediated by the subunit.

[0054] Together, BMPs and GDFs form a family of cysteine ​​knot cytokines that share the characteristic fold of the TGF-β superfamily (Rider et al. (2010) Biochem J., 429(1):1-12). Members of this family include, 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. Apart from the ability to induce bone formation that gave BMPs their name, BMPs / GDFs exhibit morphogenetic activity in the development of a wide range of tissues. BMP / GDF homo- and heterodimers interact with combinations of type I and type II receptor dimers to generate multiple possible signaling complexes that result in the activation of one of two competing sets of SMAD transcription factors. BMP / GDFs have highly specific and localized functions. They are regulated in multiple ways, including through the developmental restriction of BMP / GDF expression and the secretion of several specific BMP antagonist proteins that bind with high affinity to cytokines. Curiously, several of these antagonists also resemble TGF-β superfamily ligands.

[0055] Growth and differentiation factor-8 (GDF8) is also known as myostatin. GDF8 is a negative regulator of skeletal muscle mass and is highly expressed in developing and adult skeletal muscle. GDF8 null mutations in transgenic mice are characterized by marked hypertrophy and hyperplasia of skeletal muscle [McPherron et al. Nature (1997) 387:83-90]. Similar increases in skeletal muscle mass are evident in cattle and, particularly, in humans with naturally occurring mutations of GDF8 (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; Kambadur et al. Genome Res. (1997) 7:910-915; and Schuelke et al. (2004) N Engl J Med, 350:2682-8). Studies have also shown that muscle atrophy associated with HIV infection in humans is accompanied by an increase in GDF8 protein expression (Gonzalez-Cadavid et al., PNAS (1998) 95:14938-43). In addition, GDF8 can alter the production of muscle-specific enzymes (e.g., creatine kinase) and alter myoblast proliferation [WO 00 / 43781]. The GDF8 propeptide can non-covalently bind to the processed GDF8 domain dimer and inactivate 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 proteins that bind to and inhibit the biological activity of GDF8 and structurally related proteins include follistatin and potentially follistatin-related proteins [Gamer et al. (1999) 99) Dev. Biol., 208: 222-232].

[0056] GDF11, also known as BMP11, is a secreted protein expressed in the developing mouse tail bud, limb bud, maxillary and mandibular arches, and dorsal root ganglia [McPherron et al. (1999) Nat. Genet., 22: 260-264; and Nakashima et al. (1999) Mech. Dev., 80: 185-189]. GDF11 plays a unique role in the patterning of both mesodermal and neural tissues [Gamer et al. (1999) Dev Biol., 208:222-32]. GDF11 has been shown to be a negative regulator of chondrogenesis and myogenesis in the developing chick limb [Gamer et al. (2001) Dev Biol., 229:407-20]. Expression of GDF11 in muscle also suggests its role in regulating muscle growth similar to GDF8. In addition, the expression of GDF11 in the brain suggests that GDF11 may also have activities related to the function of the nervous system.Interestingly, GDF11 has been found to inhibit the neurogenesis of olfactory epithelium [Wu et al. (2003) Neuron., 37:197-207].Therefore, GDF11 may have in vitro and in vivo applications in the treatment of diseases such as muscle diseases and neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).

[0057] In part, this disclosure relates to the discovery that activin and / or GDF antagonists (inhibitors) treat or reduce the rate of progression and / or severity of kidney disease, and in particular treat, prevent, or reduce the rate of progression and / or severity of one or more kidney disease-related complications (e.g., kidney tissue damage, fibrosis, and / or inflammation). In some embodiments, the disclosure relates to the use of activin and / or GDF antagonists that inhibit one or more activins (e.g., activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF11, GDF8, GDF3, GDF1, nodal, ALK4, ALK5, ALK7, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, cryptic, cryptic 1B, Smad2, and Smad3, etc.) for use in treating, preventing, or reducing the rate of progression and / or severity of kidney disease, or treating, preventing, or reducing the rate of progression, frequency, and / or severity of one or more kidney disease-related complications (e.g., kidney tissue damage, fibrosis, and / or inflammation).

[0058] The terms used herein generally have their usual meaning in the art in the context of this disclosure and in the specific context in which each term is used.In order to provide practitioners with additional guidance in the description of the compositions and methods of this disclosure and their preparation and use methods, certain terms are explained below and elsewhere in this specification.The scope or meaning of any of the terms used will be clear from the specific context in which the term is used.

[0059] "Homology," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that have a "common evolutionary origin," including proteins from superfamilies in the same species and homologous proteins from different species. Such proteins (and their encoding nucleic acids) have sequence homology, including in terms of percentage identity, or by the presence of specific residues or motifs and conserved positions. However, in common usage and in this application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity, with or without reference to a common evolutionary origin.

[0060] The term "sequence similarity" in all its grammatical forms means the degree of identity or correspondence between multiple nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

[0061] "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 sequence (or nucleic acid) in the reference polypeptide (nucleotide) sequence, where identity is determined after aligning the sequences and introducing gaps as necessary to obtain the maximum sequence identity (%), and does not consider any conservative substitutions as part of the sequence identity. Alignment to determine amino acid sequence identity (%) can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of the present invention, amino acid (nucleic acid) sequence identity (%) is obtained using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0062] "Agonize," in all its grammatical forms, refers to the process of activating a protein and / or gene (e.g., by activating or amplifying gene expression of that protein or by inducing an inactive protein to become active) or increasing the activity of a protein and / or gene.

[0063] "Antagonize," in all its grammatical forms, refers to the process of inhibiting a protein and / or gene (e.g., by inhibiting or reducing gene expression of the protein or by inducing an active protein to become inactive) or reducing the activity of a protein and / or gene.

[0064] The terms "about" and "approximately" as used in reference to numerical values ​​throughout this specification and claims refer to an interval of accuracy well known and accepted by those of skill in the art. Typically, such an interval is ±10%. Alternatively, particularly in biological systems, the terms "about" and "approximately" can refer to values ​​that are within an order of magnitude (10-fold) of the given value, preferably within 5-fold, and more preferably within 2-fold. The numerical ranges disclosed herein are inclusive of the numbers defining the range.

[0065] The singular terms "a" and "an" include plural referents unless the context in which the term is used clearly contradicts. The singular and the terms "one or more" and "at least one" may be used interchangeably herein. Furthermore, "and / or" as used herein should be considered a specific disclosure of each of two or more specified features or components, with or without the others. Thus, the term "and / or" as used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" as used in a phrase such as "A, B and / or C" is intended to include each of the following embodiments: 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 (single); B (single); and C (single).

[0066] Throughout this specification, "comprises" or variations such as "comprises" or "including" are understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0067] 2. TGF-β superfamily type I receptor, type II receptor, and co-receptor polypeptides, their variants, and protein complexes In certain embodiments, the activin and / or GDF antagonist to be used according to the methods and uses disclosed herein is an ActRII polypeptide (e.g., ActRIIA or ActRIIB polypeptide), a variant thereof, or a protein complex comprising at least one ActRII polypeptide (e.g., a homodimer comprising two ActRII polypeptides or a heterodimer comprising one ActRII polypeptide and a heterologous polypeptide (e.g., ALK4)). As used herein, the term "ActRII" refers to the family of type II activin receptors. This family includes type IIA activin receptor (ActRIIA) and type IIB activin receptor (ActRIIB). The ActRII polypeptide or a protein complex comprising an ActRII polypeptide can, for example, inhibit one or more ActRII-binding ligands (e.g., activin, GDF8, GDF11, GDF3, GDF1 and nodal), ActRII receptors (e.g., ActRIIA and ActRIIB), ActRII-related type I receptors (e.g., ALK4, ALK5, ALK7, etc.) and / or ActRII-related co-receptors (e.g., crypt, cryptic, cryptic 1B, etc.). In some embodiments, the ability of the ActRII polypeptide or a protein complex comprising an ActRII polypeptide to inhibit signal transduction (Smad signal transduction) is determined, for example, in a cell-based assay, including those described herein. The ActRII polypeptide or a protein complex comprising an ActRII polypeptide can be used to treat, prevent, or reduce the rate of progression and / or severity of kidney disease or one or more complications of kidney disease, either alone or in combination with one or more additional adjunctive therapies or active agents.

[0068] As used herein, the term "ActRIIB" refers to the family of activin type IIB receptor (ActRIIB) proteins from any species and the variants derived from such ActRIIB proteins by mutagenesis or other modifications.In this specification, reference to ActRIIB is understood as a reference to any one of the currently identified forms.Members of the ActRIIB family are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0069] The term "ActRIIB polypeptide" includes any naturally occurring polypeptide of the ActRIIB family members, as well as any variant thereof (including mutants, fragments, fusions, and peptidomimetics forms) that retains useful activity. Examples of such mutant ActRIIB polypeptides are provided throughout this disclosure, as well as in International Publication Nos. 2006 / 012627, 2008 / 097541, 2010 / 151426, and 2011 / 020045, which are incorporated herein by reference in their entirety. The amino acid numbering for all ActRIIB-related polypeptides described herein is based on the numbering of the human ActRIIB precursor protein sequence (SEQ ID NO: 1; NCBI database accession number NP_001097.2), unless specifically stated otherwise. Another ActRIIB mutant containing an arginine to alanine substitution at position 64 is shown in SEQ ID NO: 2.

[0070] The processed extracellular ActRIIB polypeptide sequence is represented in SEQ ID NO: 3. The alternative A64 form of the processed extracellular ActRIIB polypeptide sequence is represented in SEQ ID NO: 4. The C-terminal "tail" of the extracellular domain is indicated by a single underline in both sequences.

[0071] In some embodiments, the protein can be produced containing an "SGR..." sequence at the N-terminus. The "tail" deleted sequence (Δ15 sequence) is shown in SEQ ID NOs: 5 and 6, which represent the wild type and R64A mutant, respectively.

[0072] The nucleic acid sequence encoding the human ActRIIB precursor protein is shown as SEQ ID NO:7, which represents nucleotides 25-1560 of the Genbank reference sequence NM_001106.3, which encodes amino acids 1-513 of the ActRIIB precursor. The sequence shown can be modified to provide an arginine at position 64, and an alanine instead. The signal sequence is underlined.

[0073] A nucleic acid sequence encoding the processed extracellular human ActRIIB polypeptide (SEQ ID NO: 3) is shown in SEQ ID NO: 8. The codon for arginine at position 64 of this sequence can instead be replaced with a codon for alanine.

[0074] An alignment of the amino acid sequences of the human ActRIIB extracellular domain and the human ActRIIA extracellular domain is shown in FIG. 1. This alignment shows the amino acid residues in both receptors that are believed to directly contact the ActRII ligand. For example, the composite ActRII structure showed that the ActRIIB ligand-binding pocket is defined, in part, by residues Y31, N33, N35, L38-T41, E47, E50, Q53-K55, L57, H58, Y60, S62, K74, W78-N83, Y85, R87, A92, and E94-F101. It is predicted that conservative mutations will be tolerated at these positions.

[0075] In addition, ActRIIB is highly conserved in vertebrates, with large stretches of the extracellular domain being completely conserved. For example, FIG. 2 shows multiple sequence alignments of the human ActRIIB extracellular domain compared to various ActRIIB orthologs. Many of the ligands that bind to ActRIIB are also highly conserved. Thus, from these alignments, it is possible to predict the important amino acid positions in the ligand-binding domain that are important for normal ActRIIB ligand-binding activity, and to predict the amino acid positions that will tolerate substitution without significantly changing normal ActRIIB ligand-binding activity. Thus, an active human ActRIIB mutant polypeptide that is useful according to the methods disclosed herein can include one or more amino acids at the corresponding positions from the sequence of another vertebrate ActRIIB, or can include similar residues as those in human or other vertebrate sequences. Without being meant to be limiting, the following examples illustrate the present approach to define active ActRIIB mutants. L46 in the human extracellular domain is a valine in Xenopus ActRIIB, meaning this position can be altered and optionally altered to another hydrophobic residue such as V, I or F, or a non-polar residue such as A. E52 in the human extracellular domain is a K in Xenopus, indicating that this site can tolerate a wide range of changes including polar residues such as E, D, K, R, H, S, T, P, G, Y and preferably A. T93 in the human extracellular domain is a K in Xenopus, indicating that this position tolerates wide structural diversity, with polar residues such as S, K, R, E, D, H, G, P, G and Y being preferred. F108 in the human extracellular domain is a Y in Xenopus, therefore Y or other hydrophobic groups such as I, V or L should be tolerated. E111 in the human extracellular domain is K in Xenopus, indicating that charged residues including D, R, K and H, as well as Q and N, would be tolerated at this position.R112 in the human extracellular domain is a K in Xenopus, indicating that acidic residues, including R and H, are tolerated at this position. A at position 119 in the human extracellular domain is less conserved, appearing as a P in rodents and a V in Xenopus, meaning that essentially any amino acid should be tolerated at this position.

[0076] Furthermore, ActRII proteins have been characterized in the art for structural and 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; and U.S. Patent Nos. 7,709,605, 7,612,041, and 7,842,663]. In addition to the teachings herein, these references provide ample guidance on how to create ActRIIB mutants that retain one or more desired activities (e.g., ligand binding activity).

[0077] For example, a defined structural motif known as the three-finger toxic fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor (Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870). Thus, the core ligand-binding domain of human ActRIIB as defined by the outermost of these conserved cysteines corresponds to positions 29-109 of SEQ ID NO:1 (ActRIIB precursor). The structurally less defined amino acids adjacent to these cysteine-defining 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, or 28 residues at the N-terminus, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 residues at the C-terminus, without necessarily altering ligand binding. Exemplary ActRIIB extracellular domains for N- and / or C-terminal truncations include SEQ ID NOs: 3, 4, 5, and 6.

[0078] Attisano et al. showed that deletion of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduced the affinity of the receptor for activin. An ActRIIB-Fc fusion protein containing amino acids 20-119 of SEQ ID NO:1 herein, "ActRIIB(20-119)-Fc," has reduced binding to GDF11 and activin compared to ActRIIB(20-134)-Fc, which contains the proline knot region and the complete juxtamembrane domain (see, e.g., U.S. Patent No. 7,842,663). However, the ActRIIB(20-129)-Fc protein retains similar, but somewhat reduced, activity compared to the wild type, despite the disruption of the proline knot region.

[0079] That is, ActRIIB extracellular domains terminating at amino acids 134, 133, 132, 131, 130 and 129 (with respect to SEQ ID NO: 1) are all predicted to be active, but constructs terminating at amino acids 134 or 133 may be the most active. Similarly, mutations at any of residues 129-134 (with respect to SEQ ID NO: 1) are predicted to alter ligand binding affinity by a large margin. In support of this, it is known in the art that mutations at P129 and P130 (with respect to SEQ ID NO: 1) do not substantially reduce ligand binding. Thus, the ActRIIB polypeptides of the present disclosure can terminate as early as amino acid 109 (the last cysteine), however, forms terminating at 109-119 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are predicted to have reduced ligand binding. Amino acid 119 (with respect to SEQ ID NO:1 of the present disclosure) is less conserved and therefore is easily modified or truncated. ActRIIB polypeptides terminating at or after amino acid 128 (with respect to SEQ ID NO:1) should retain ligand binding activity. ActRIIB polypeptides terminating at or between amino acids 119-127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, or 127) (with respect to SEQ ID NO:1) will have moderate binding ability. Any of these forms may be desirable for use depending on clinical or experimental conditions.

[0080] At the N-terminus of ActRIIB, proteins starting at or before amino acid 29 (with respect to SEQ ID NO:1) are predicted to retain ligand-binding activity. Amino acid 29 represents the first cysteine. An alanine to asparagine mutation at position 24 (with respect to SEQ ID NO:1) introduces an N-linked glycosylation sequence without substantially affecting ligand binding (U.S. Patent No. 7,842,663). This confirms that mutations in the region between the signal cleavage peptide and the cysteine ​​bridge region (corresponding to amino acids 20-29) are well tolerated. In particular, ActRIIB polypeptides starting at positions 20, 21, 22, 23, and 24 (with respect to SEQ ID NO:1) should generally retain ligand-binding activity, and ActRIIB polypeptides starting at positions 25, 26, 27, 28, and 29 (with respect to SEQ ID NO:1) are also predicted to retain ligand-binding activity. Surprisingly, it has been demonstrated that ActRIIB constructs starting at positions 22, 23, 24, or 25 appear to have the strongest activity (eg, US Pat. No. 7,842,663).

[0081] Taken together, a general formula for an active portion (e.g., a ligand-binding portion) of ActRIIB includes amino acids 29 to 109 of SEQ ID NO: 1. Thus, an ActRIIB polypeptide can begin, for example, at a residue corresponding to any one of amino acids 20 to 29 of SEQ ID NO: 1 (e.g., begin at any one of amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) and end at a position corresponding to any one of amino acids 109 to 134 of SEQ ID NO: 1 (e.g., begin at amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 17 In one embodiment, the amino acid sequence of ActRIIB can 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 ActRIIB (terminating at any one of: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134). Other examples include a sequence starting at positions 20 to 29 (e.g., any one of positions 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) or a sequence starting at positions 21 to 29 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO:1 and continuing at positions 119 to 134 (e.g., positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO:1. or at any one of positions 129, 130, 131, 132, or 133).Other examples include a nucleotide sequence starting at positions 20 to 24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21 to 24 (e.g., any one of positions 21, 22, 23, or 24), or 22 to 25 (e.g., any one of positions 22, 22, 23, or 25) of SEQ ID NO:1 and not including positions 109 to 134 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or terminating at any one of positions 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-134 (e.g., any one of positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), or 129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134). Variants within these ranges are also contemplated, particularly those comprising, consisting essentially of, or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ ID NO:1.

[0082] The mutations described herein can be combined in various ways. In some embodiments, the ActRIIB mutant comprises no more than 1, 2, 5, 6, 7, 8, 9, 10 or 15 conservative amino acid changes in the ligand-binding pocket, and optionally 0, 1 or more non-conservative changes at positions 40, 53, 55, 74, 79 and / or 82 in the ligand-binding pocket. Sites outside the binding pocket where diversity may be particularly well tolerated include the amino- and carboxy-termini of the extracellular domain (as described above), as well as positions 42-46 and 65-73 (with respect to SEQ ID NO:1). An asparagine to alanine change at position 65 (N65A) does not appear to reduce ligand binding in the R64 background (U.S. Patent No. 7,842,663). This change likely removes glycosylation at N65 in the A64 background, thus demonstrating that significant changes in this region will be tolerated. While the change R64A is poorly tolerated, R64K is well tolerated, i.e., another basic residue such as H can be tolerated at position 64 (U.S. Patent No. 7,842,663). In addition, the results of mutagenesis programs described in the art indicate that there are often amino acid positions in ActRIIB that are beneficial to conserve. With respect to SEQ ID NO:1, these include positions 80 (acidic or hydrophobic amino acids), 78 (hydrophobic, especially tryptophan), 37 (acidic, especially aspartic acid or glutamic acid), 56 (basic amino acids), and 60 (hydrophobic amino acids, especially phenylalanine or tyrosine). Thus, the present disclosure provides a framework of amino acids that can be conserved in ActRIIB polypeptides. Other positions that may be desirable to conserve are as follows: positions 52 (acidic amino acids), 55 (basic amino acids), 81 (acidic), and 98 (polar or charged, especially E, D, R, or K), all with respect to SEQ ID NO:1.

[0083] It has been previously demonstrated that the addition of additional N-linked glycosylation sites (NXS / T) to the ActRIIB extracellular domain is well tolerated (see, e.g., U.S. Pat. No. 7,842,663). Thus, NXS / T sequences can be generally introduced into the ActRIIB polypeptides of the present disclosure at positions outside the ligand-binding pocket as defined in FIG. 1. Particularly suitable sites for the introduction of non-endogenous NXS / T sequence sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134, or 129-134 (with respect to SEQ ID NO: 1). NXS / T sequences can also be introduced into the linker between ActRIIB and the Fc domain or other fusion component, as well as optionally into the fusion component itself. Such sites can be introduced with minimal effort by introducing an N at the correct position relative to an existing S or T, or by introducing an S or T at a position corresponding to an existing N. In other words, desirable modifications that will generate N-linked glycosylation sites are the following modifications: A24N, R64N, S67N (possibly in combination with the N65A modification), E105N, R112N, G120N, E123N, P129N, A132N, R112S and R112T (with respect to SEQ ID NO: 1). Any S predicted to be glycosylated can be modified to T without generating an immunogenic site due to the protection afforded by glycosylation. Similarly, any T predicted to be glycosylated can be modified to S. Thus, modifications S67T and S44T (with respect to SEQ ID NO: 1) are contemplated. Similarly, in the A24N mutant, the S26T modification can be used. Thus, the ActRIIB polypeptides of the present disclosure can be mutants having one or more additional non-intrinsic N-linked glycosylation consensus sequences as described above.

[0084] In certain embodiments, the activin and / or GDF antagonist that is the subject of the method and use disclosed herein is ActRIIB polypeptide (including its fragment and functional variant) and protein complex that comprises at least one ActRIIB polypeptide (for example, homodimer that comprises two ActRIIB polypeptides or heterodimer that comprises one ActRIIB polypeptide and a heterologous polypeptide (for example, ALK4).Preferably, the ActRIIB polypeptide is soluble (for example, comprises the extracellular domain of ActRIIB). In some embodiments, the ActRIIB polypeptide antagonizes the activity (e.g., Smad signaling) of one or more activin and / or GDF ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin E), GDF3, GDF1, nodal, ActRIIA, ActRIIB, ALK4, ALK5, ALK7, cryptic, cryptic 1B, Smad2, and Smad3). Thus, in some embodiments, the ActRIIB polypeptide binds to one or more activin and / or GDF ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF3, GDF1, nodal, ActRIIA, ALK4, ALK5, ALK7, cryptic, cryptic 1B, Smad2, and Smad3, etc.). In some embodiments, an ActRIIB polypeptide of the disclosure comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIB beginning at residues corresponding to amino acids 20-29 of SEQ ID NO:1 and ending at positions corresponding to amino acids 109-134 of SEQ ID NO:1.In some embodiments, an ActRIIB 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 amino acids 29-109 of SEQ ID NO: 1. In certain embodiments, an ActRIIB polypeptide of the disclosure 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 amino acids 25-131 of SEQ ID NO: 1. In some embodiments, an ActRIIB polypeptide of the present disclosure comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 181, 182, 184, 187, 188, 189, 190, 192, 193, 196, 197, 198, 199, 201, 205, and 206. In some embodiments, an ActRIIB polypeptide of the present disclosure comprises an ActRIIB polypeptide in which the position corresponding to L79 of SEQ ID NO: 1 is not an acidic amino acid (i.e., not a naturally occurring acidic amino acid D or E or an artificial acidic amino acid residue).

[0085] In certain embodiments, the present disclosure relates to ActRIIA polypeptide.As used herein, the term "ActRIIA" refers to the family of activin type IIA receptor (ActRIIA) proteins from any species and the variants derived from such ActRIIA proteins by mutagenesis or other modifications.In this specification, reference to ActRIIA is understood to refer to any one of the currently identified forms.Members of the ActRIIA family are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0086] The term "ActRIIA polypeptide" includes polypeptides including any naturally occurring polypeptide of the ActRIIA family members, as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. Examples of such mutant ActRIIA polypeptides are provided throughout this disclosure, as well as in International Publication Nos. WO 2006 / 012627 and WO 2007 / 062188, which are incorporated herein by reference in their entireties. The human ActRIIA precursor protein sequence is represented by SEQ ID NO:9 or SEQ ID NO:10.

[0087] The processed extracellular human ActRIIA polypeptide sequence is represented in SEQ ID NO: 11. The nucleic acid sequence encoding the processed extracellular ActRIIA polypeptide has the sequence of SEQ ID NO:14. The sequence having the extracellular domain with the C-terminal "tail" deleted (Δ15 sequence) is represented in SEQ ID NO:12.

[0088] The nucleic acid sequence encoding the human ActRIIA precursor protein (SEQ ID NO:9) is shown below (SEQ ID NO:13) and corresponds to nucleotides 159-1700 of the Genbank reference sequence NM_001616.4. The signal sequence is underlined.

[0089] ActRIIA is highly conserved in vertebrates, and large stretches of the extracellular domain are completely conserved. For example, FIG. 3 shows multiple sequence alignment of the human ActRIIA extracellular domain compared to various ActRIIA orthologs. Many of the ligands that bind to ActRIIA are also highly conserved. Thus, from these alignments, it is possible to predict the important amino acid positions in the ligand-binding domain that are important for normal ActRIIA ligand binding activity, as well as predict the amino acid positions that may tolerate substitution without significantly changing normal ActRIIA ligand binding activity. Thus, the active human ActRIIA mutant polypeptides that are useful according to the methods disclosed herein can include one or more amino acids at the corresponding positions from the sequence of another vertebrate ActRIIA, or can include similar residues as those in human or other vertebrate sequences.

[0090] Without being meant to be limiting, the following example illustrates this approach to defining active ActRIIA variants. As shown in Figure 3, F13 in the human extracellular domain is Y in Ovis aries (SEQ ID NO: 18), Gallus gallus (SEQ ID NO: 21), Bos Taurus (SEQ ID NO: 22), Tyto alba (SEQ ID NO: 23), and Myotis davidii (SEQ ID NO: 24) ActRIIA, indicating that aromatic residues including F, W, and Y are tolerated at this position. Q24 in the human extracellular domain is R in bovine ActRIIA, indicating that charged residues including D, R, K, H, and E would be tolerated at this position. S95 in the human extracellular domain is F in chicken and barn owl ActRIIA, indicating that a wide range of modifications can be tolerated at this site, including polar residues such as E, D, K, R, H, S, T, P, G, Y, and possibly hydrophobic residues such as L, I, or F. E52 in the human extracellular domain is D in ovine ActRIIA, indicating that acidic residues, including D and E, are tolerated at this position. P29 in the human extracellular domain is less conserved and is S in ovine ActRIIA and L in Myotis davidii ActRIIA, indicating that essentially any amino acid should be tolerated at this position.

[0091] Furthermore, as mentioned above, ActRII proteins have been characterized in the art for their 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; and U.S. Patent Nos. 7,709,605, 7,612,041, and 7,842,663]. In addition to the teachings herein, these references provide ample guidance on how to create ActRII mutants that retain one or more desired activities (e.g., ligand binding activity).

[0092] For example, a defined structural motif known as the three-finger toxic fold is important for ligand binding by type I and type II receptors and is formed by conserved cysteine ​​residues located at various positions within the extracellular domain of each monomeric receptor (Greenwald et al. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett 586:1860-1870). Thus, the core ligand-binding domain of human ActRIIA, as defined by the outermost of these conserved cysteines, corresponds to positions 30-110 of SEQ ID NO:9 (ActRIIA precursor). Thus, the structurally less defined amino acids adjacent to these cysteine-defining core sequences can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 residues at the N-terminus and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues at the C-terminus without necessarily altering ligand binding. Exemplary ActRIIA extracellular domain truncations include SEQ ID NO:12.

[0093] Thus, a general formula for an active portion (e.g., ligand binding) of ActRIIA is a polypeptide comprising, consisting essentially of, or consisting of amino acids 30-110 of SEQ ID NO: 9. Thus, 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 a portion of ActRIIA, e.g., beginning with a residue corresponding to any one of amino acids 21-30 of SEQ ID NO: 9 and ending at a position corresponding to any one of amino acids 110-135 of SEQ ID NO: 9. Other examples include constructs starting at a position selected from amino acids 21-30, 22-30, 23-30, 24-30 of SEQ ID NO: 9, and terminating at a position selected from amino acids 111-135, 112-135, 113-135, 120-135, 130-135, 111-134, 111-133, 111-132, or 111-131 of SEQ ID NO: 9. Variants within this range are also contemplated, particularly those that contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding portion of SEQ ID NO: 9. Thus, in some embodiments, an ActRIIA polypeptide can include a polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO: 9. Optionally, an ActRIIA polypeptide includes 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: 9 and includes no more than 1, 2, 5, 10, or 15 conservative amino acid changes in the ligand binding pocket.

[0094] In certain embodiments, the activin and / or GDF antagonists that are the subject of the methods and uses disclosed herein are ActRIIA polypeptides (including fragments and functional variants thereof) and protein complexes that contain at least one ActRIIA polypeptide (e.g., homodimers that contain two ActRIIA polypeptides or heterodimers that contain one ActRIIA polypeptide and a heterologous polypeptide (e.g., ALK4).Preferably, the ActRIIA polypeptide is soluble (e.g., the extracellular domain of ActRIIA). In some embodiments, the ActRIIA polypeptide inhibits one or more activin and / or GDF ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF3, GDF1, nodal, ActRIIB, ALK4, ALK5, ALK7, cryptic, cryptic 1B, Smad2, and Smad3, etc.) (e.g., Smad signaling). In some embodiments, the ActRIIA polypeptide binds to one or more activin and / or GDF ligands (e.g., GDF11, GDF8, activin (activin A, activin B, activin AB, activin C, activin AC, activin BC, activin E, activin AE, and / or activin BE), GDF3, GDF1, nodal, ActRIIB, ALK4, ALK5, ALK7, cryptic, cryptic 1B, Smad2, and Smad3, etc.). In some embodiments, an ActRIIA polypeptide of the disclosure comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIA beginning with residues corresponding to amino acids 21-30 of SEQ ID NO:9 and ending at a position corresponding to amino acids 110-135 of SEQ ID NO:9.In some embodiments, 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 amino acids 30-110 of SEQ ID NO: 9. In certain embodiments, 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 amino acids 21-135 of SEQ ID NO: 9. In some embodiments, the ActRIIA polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 9, 10, 11, 12, 15, 18-24, 177, 178, and 180.

[0095] In certain aspects, the present disclosure relates to heteromultimers comprising BMPRII polypeptides.As used herein, the term "BMPRII" refers to the family of bone morphogenetic protein receptor type II (BMPRII) proteins from any species and variants derived from such BMPRII proteins by mutagenesis or other modifications.Reference herein to BMPRII is understood to refer to any one of the currently identified forms.BMPRII family members are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0096] The term "BMPRII polypeptide" includes polypeptides including any naturally occurring polypeptide of the BMPRII family members as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all BMPRII-related polypeptides described herein is based on the numbering of the human BMPRII precursor protein sequence (SEQ ID NO: 34, NCBI Reference Sequence NP_001195.2), unless specifically stated otherwise. In SEQ ID NO: 34, the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold. The nucleic acid sequence encoding the BMPRII precursor protein is shown in SEQ ID NO: 35, which is as per nucleotides 1149-4262 of Genbank Reference Sequence NM_001204.6. The processed extracellular BMPRII polypeptide sequence is represented in SEQ ID NO: 36, which can be encoded by the nucleic acid sequence of SEQ ID NO: 37. An alternative isoform of BMPRII, isoform 2 (GenBank: AAA86519.1), is represented in SEQ ID NO: 38 (signal peptide is single underlined, extracellular domain is bolded) and can be encoded by the nucleic acid sequence of SEQ ID NO: 39 (corresponding to nucleotides 163-1752 of Genbank reference sequence U25110.1, signal sequence is underlined). The processed extracellular BMPRII polypeptide sequence (isoform 2) is represented in SEQ ID NO: 40 and can be encoded by the nucleic acid sequence of SEQ ID NO: 41.

[0097] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one BMPRII polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the BMPRII polypeptide for use according to the disclosed invention (e.g., heteromultimers comprising BMPRII polypeptides and uses thereof) is soluble (e.g., the extracellular domain of BMPRII). In other preferred embodiments, the BMPRII polypeptide for use according to the disclosed invention binds to and / or inhibits (antagonizes) the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, the heteromultimers of the present disclosure comprise at least one BMPRII 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: 34, 36, 38, or 40. In some embodiments, the heteromultimer of the present disclosure begins at any one of amino acids 27-34 (e.g., amino acid residues 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO: 34 and includes any one of amino acids 123-150 (e.g., amino acid residues 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344 In some embodiments, the heteromultimer of the present disclosure comprises at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of SEQ ID NO:34, SEQ ID NO:37, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150. In some embodiments, the heteromultimer of the present disclosure comprises at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 27-123 of SEQ ID NO:34.In some embodiments, heteromultimers of the disclosure comprise at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 27-150 of SEQ ID NO: 34. In some embodiments, heteromultimers of the disclosure comprise at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-123 of SEQ ID NO: 34. In some embodiments, heteromultimers of the disclosure include at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-150 of SEQ ID NO: 34. In some embodiments, heteromultimers of the disclosure include at least one BMPRII polypeptide that begins at any one of amino acids 27-34 (e.g., amino acid residues 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO: 38 and begins at any one of amino acids 123-150 (e.g., amino acid residues 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 2 In some embodiments, the heteromultimer of the present disclosure comprises at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of SEQ ID NO:37, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150. In some embodiments, the heteromultimer of the present disclosure comprises at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 27-123 of SEQ ID NO:38.In some embodiments, heteromultimers of the disclosure comprise at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 27-150 of SEQ ID NO: 38. In some embodiments, heteromultimers of the disclosure comprise at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-123 of SEQ ID NO: 38. In some embodiments, heteromultimers of the disclosure comprise at least one BMPRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-150 of SEQ ID NO:38.

[0098] In certain aspects, the present disclosure relates to heteromultimers comprising MISRII polypeptides.As used herein, the term "MISRII" refers to the family of Mullerian inhibitory receptor type II (MISRII) proteins from any species and the variants derived from such MISRII proteins by mutagenesis or other modifications.Any reference to MISRII herein is understood to refer to any one of the currently identified forms.Members of the MISRII family are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0099] The term "MISRII polypeptide" includes polypeptides including any naturally occurring polypeptide of the MISRII family members as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all MISRII-related polypeptides described herein is based on the numbering of the human MISRII precursor protein sequence (SEQ ID NO: 42, NCBI Reference Sequence NP_065434.1), unless specifically stated otherwise. In SEQ ID NO: 42, the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold. The nucleic acid sequence encoding the MISRII precursor protein is shown in SEQ ID NO: 43, as per nucleotides 81-1799 of Genbank Reference Sequence NM_020547.2. The processed extracellular MISRII polypeptide sequence (isoform 1) is represented in SEQ ID NO: 44, which can be encoded by the nucleic acid sequence of SEQ ID NO: 45. An alternative isoform of the human MISRII precursor protein sequence, isoform 2 (NCBI Reference Sequence NP_001158162.1), is depicted in SEQ ID NO: 46 (the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold), which can be encoded by the nucleic acid sequence of SEQ ID NO: 47 (corresponding to nucleotides 81-1514 of Genbank Reference Sequence NM_001164690.1). The processed extracellular MISRII polypeptide sequence (isoform 2) is 100% identical to the processed extracellular MISRII polypeptide sequence (isoform 1) (i.e., it has the amino acid sequence of SEQ ID NO: 44, which can be encoded by the nucleic acid sequence of SEQ ID NO: 45). An alternative isoform of the human MISRII precursor protein sequence, isoform 3 (NCBI Reference Sequence NP_001158163.1), is depicted in SEQ ID NO: 48 (the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold), which can be encoded by the nucleic acid sequence of SEQ ID NO: 49 (corresponding to nucleotides 81 to 1514 of Genbank Reference Sequence NM_001164691.1). The signal sequence is underlined.The processed extracellular MISRII polypeptide sequence (isoform 3) is 100% identical to the processed extracellular MISRII polypeptide sequence (isoform 1) (i.e., it has the amino acid sequence of SEQ ID NO: 44, which can be encoded by the nucleic acid sequence of SEQ ID NO: 45).

[0100] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one MISRII polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the MISRII polypeptide (e.g., heteromultimers comprising MISRII polypeptides and uses thereof) for use according to the disclosed invention is soluble (e.g., the extracellular domain of MISRII). In other preferred embodiments, the MISRII polypeptide for use according to the disclosed invention binds to and / or inhibits (antagonizes) the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, the heteromultimers of the present disclosure comprise at least one MISRII 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: 42, 44, 46, or 48. In some embodiments, the heteromultimer of the disclosure begins at any one of amino acids 17-24 (e.g., amino acid residues 17, 18, 19, 20, 21, 22, 23, or 24) of SEQ ID NO:42 and includes any one of amino acids 116-149 (e.g., amino acid residues 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, In some embodiments, the present invention further comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of the following sites: In some embodiments, the heteromultimer of the present disclosure comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 17-116 of SEQ ID NO:42.In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 17-149 of SEQ ID NO: 42. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-116 of SEQ ID NO: 42. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-149 of SEQ ID NO: 42. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that begins at any one of amino acids 17-24 (e.g., amino acid residues 17, 18, 19, 20, 21, 22, 23, or 24) of SEQ ID NO: 46 and begins at any one of amino acids 116-149 (e.g., amino acid residues 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 In some embodiments, the present invention further comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of the following sites: In some embodiments, the heteromultimer of the present disclosure comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 17-116 of SEQ ID NO:46.In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 17-149 of SEQ ID NO: 46. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-116 of SEQ ID NO: 46. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-149 of SEQ ID NO: 46. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that begins at any one of amino acids 17-24 (e.g., amino acid residues 17, 18, 19, 20, 21, 22, 23, or 24) of SEQ ID NO: 42 and begins at any one of amino acids 116-149 (e.g., amino acid residues 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174 In some embodiments, the present invention further comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of the following sites: In some embodiments, the heteromultimer of the present disclosure comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 17-116 of SEQ ID NO:48.In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 17-149 of SEQ ID NO: 48. In some embodiments, heteromultimers of the disclosure comprise at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-116 of SEQ ID NO: 48. In some embodiments, the heteromultimer of the present disclosure comprises at least one MISRII polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 24-149 of SEQ ID NO:48.

[0101] In certain aspects, the present disclosure relates to heteromultimers comprising ALK1 polypeptide.As used herein, the term "ALK1" refers to the family of activin receptor-like kinase-1 proteins from any species and the variants derived from such ALK1 proteins by mutagenesis or other modifications.Any reference to ALK1 herein is understood to refer to any one of the currently identified forms.ALK1 family members are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0102] The term "ALK1 polypeptide" includes polypeptides including any naturally occurring polypeptide of the ALK1 family members as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all ALK1-related polypeptides described herein is based on the numbering of the human ALK1 precursor protein sequence (SEQ ID NO: 52, NCBI Reference Sequence NP_000011.2), unless specifically stated otherwise. In SEQ ID NO: 52, the signal peptide is indicated by a single underline and the extracellular domain is indicated by bold. The nucleic acid sequence encoding the human ALK1 precursor protein is shown in SEQ ID NO: 53, which is as per nucleotides 284-1792 of Genbank Reference Sequence NM_000020.2. The processed extracellular ALK1 polypeptide sequence is represented in SEQ ID NO: 54, which can be encoded by the nucleic acid sequence of SEQ ID NO: 55.

[0103] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK1 polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the ALK1 polypeptide (e.g., heteromultimers comprising ALK1 polypeptides and their uses) for use according to the disclosed invention is soluble (e.g., the extracellular domain of ALK1). In other preferred embodiments, the ALK1 polypeptide for use according to the disclosed invention binds to and / or inhibits (antagonizes) the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK1 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:52 or 54. In some embodiments, the heteromultimer of the disclosure begins at any one of amino acids 22-34 (e.g., amino acid residues 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO:52 and includes any one of amino acids 95-118 (e.g., amino acid residues 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 221, 222, 223, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO:52 and includes any one of amino acids 95-118 (e.g., amino acid residues 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 220, 230, 231, 232, 233, 234, 235, 236, 237, 238, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 251, 252, 253, 254, In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an ALK1 polypeptide that terminates at any one of SEQ ID NO:52 (SEQ ID NO:52, S ...In some embodiments, heteromultimers of the disclosure comprise at least one ALK1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 22-118 of SEQ ID NO: 52. In some embodiments, heteromultimers of the disclosure comprise at least one ALK1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34-95 of SEQ ID NO:52. In some embodiments, heteromultimers of the present disclosure comprise at least one ALK1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 34 to 118 of SEQ ID NO:52.

[0104] In certain aspects, the present disclosure relates to heteromultimers comprising ALK4 polypeptide.As used herein, the term "ALK4" refers to the family of activin receptor-like kinase-4 proteins from any organism and the mutants derived from such ALK4 proteins by mutagenesis or other modifications.In this specification, reference to ALK4 is understood to refer to any one of the currently identified forms.ALK4 family members are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0105] The term "ALK4 polypeptide" includes polypeptides including any naturally occurring polypeptide of the ALK4 family members as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all ALK4-related polypeptides described herein is based on the numbering of the human ALK4 precursor protein sequence (SEQ ID NO: 56, NCBI Reference Sequence NP_004293), unless specifically stated otherwise. In SEQ ID NO: 56, the signal peptide is indicated by a single underline and the extracellular domain is indicated in bold. The nucleic acid sequence encoding the ALK4 precursor protein is shown in SEQ ID NO: 57, which corresponds to nucleotides 78-1592 of the Genbank Reference Sequence NM_004302.4. The signal sequence is underlined and the extracellular domain is indicated in bold. The processed extracellular human ALK4 polypeptide sequence is represented in SEQ ID NO: 58, which can be encoded by the nucleic acid sequence of SEQ ID NO: 59. An alternative isoform of the human ALK4 precursor protein sequence, Isoform C (NCBI Reference Sequence NP_064733.3), is represented in SEQ ID NO: 60, with the signal peptide indicated by a single underline and the extracellular domain indicated in bold. The nucleic acid sequence encoding the ALK4 precursor protein (Isoform C) is represented in SEQ ID NO: 61, which corresponds to nucleotides 78-1715 of Genbank Reference Sequence NM_020328.3. The processed extracellular ALK4 polypeptide sequence (Isoform C) is 100% identical to the corresponding processed extracellular ALK4 polypeptide sequence (i.e., has the amino acid sequence of SEQ ID NO: 58, which can be encoded by the nucleic acid sequence of SEQ ID NO: 59).

[0106] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide (including fragments, functional variants, and modified forms thereof).Preferably, the ALK4 polypeptide for use according to the disclosed invention (e.g., heteromultimers comprising ALK4 polypeptides and their uses) is soluble (e.g., the extracellular domain of ALK4).In other preferred embodiments, the ALK4 polypeptide for use according to the disclosed invention binds to and / or inhibits (antagonizes) the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands.In some embodiments, the heteromultimers of the present 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:56, 58, or 60. In some embodiments, the heteromultimer of the disclosure begins at any one of amino acids 24-34 (e.g., amino acid residues 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO:56 and includes any one of amino acids 101-126 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 In some embodiments, the heteromultimers of the present 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 an ALK4 polypeptide that terminates at any one of SEQ ID NO:56, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126. In some embodiments, the heteromultimers of the present 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 amino acids 24-101 of SEQ ID NO:56.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 amino acids 24-126 of SEQ ID NO: 56. 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 amino acids 34-101 of SEQ ID NO:56. 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 amino acids 34-126 of SEQ ID NO: 56. In some embodiments, heteromultimers of the disclosure comprise at least one ALK4 polypeptide that begins at any one of amino acids 24-34 (e.g., amino acid residues 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQ ID NO: 83 and begins 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, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 30, 31, 32, 33, or 34) of SEQ ID NO: 83 and begins 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 In some embodiments, the heteromultimers of the present 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 an ALK4 polypeptide that terminates at any one of SEQ ID NO:60, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126. In some embodiments, the heteromultimers of the present 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 amino acids 24-101 of SEQ ID NO:60.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 amino acids 24-126 of SEQ ID NO: 60. 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 amino acids 34-101 of SEQ ID NO: 60. In some embodiments, the heteromultimers of the present 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 amino acids 34 to 126 of SEQ ID NO:60.

[0107] In certain aspects, the present disclosure relates to heteromultimers comprising ALK5 polypeptide.As used herein, the term "ALK5" refers to the family of activin receptor-like kinase-5 proteins from any species and the mutants derived from such ALK5 proteins by mutagenesis or other modifications.Any reference to ALK5 herein is understood to refer to any one of the currently identified forms.ALK5 family members are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0108] The term "ALK5 polypeptide" includes polypeptides including any naturally occurring polypeptide of the ALK5 family members as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all ALK5-related polypeptides described herein is based on the numbering of the human ALK5 precursor protein sequence (SEQ ID NO: 62, NCBI Reference Sequence NP_004603.1), unless specifically stated otherwise. In SEQ ID NO: 62, the signal peptide is indicated by a single underline and the extracellular domain is indicated by bold. The nucleic acid sequence encoding the ALK5 precursor protein is shown in SEQ ID NO: 63, which corresponds to nucleotides 77-1585 of Genbank Reference Sequence NM_004612.2. The processed extracellular ALK5 polypeptide sequence is represented in SEQ ID NO: 64, which can be encoded by the nucleic acid sequence of SEQ ID NO: 65. An alternative isoform of the human ALK5 precursor protein sequence, isoform 2 (NCBI Reference Sequence XP_005252207.1), is represented in SEQ ID NO: 66 (the signal peptide is single underlined and the extracellular domain is in bold). The nucleic acid sequence encoding the human ALK5 precursor protein (isoform 2) is represented in SEQ ID NO: 67 (the signal peptide is underlined and the extracellular domain is in bold), which corresponds to nucleotides 77 to 1597 of Genbank Reference Sequence XM_005252150.1. The processed extracellular ALK5 polypeptide sequence (isoform 2) is depicted in SEQ ID NO:68, which can be encoded by the nucleic acid sequence of SEQ ID NO:69.

[0109] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide (including fragments, functional variants, and modified forms thereof).Preferably, the ALK5 polypeptide (e.g., heteromultimers comprising ALK5 polypeptides and their uses) for use according to the disclosed invention is soluble (e.g., the extracellular domain of ALK5).In other preferred embodiments, the ALK5 polypeptide for use according to the disclosed invention binds to one or more TGF-β superfamily ligands and / or inhibits (antagonizes) their activity (e.g., Smad signaling).In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK5 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:62, 64, 66, or 68. In some embodiments, the heteromultimers of the disclosure begin at any one of amino acids 25-36 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) of SEQ ID NO:62 and begin 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, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 17 In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an ALK5 polypeptide that terminates at any one of SEQ ID NO:62, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126. In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-101 of SEQ ID NO:62.In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-126 of SEQ ID NO: 62. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 36-101 of SEQ ID NO:62. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 36-126 of SEQ ID NO: 62. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that begins at any one of amino acids 25-36 (e.g., amino acid residues 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) of SEQ ID NO: 66 and begins at any one of amino acids 101-130 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 30, 31, 32, 33, 34, 35, or 36) of SEQ ID NO: 66 and begins at any one of amino acids 101-130 (e.g., amino acid residues 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 1 In some embodiments, the ALK5 polypeptide comprises at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of the following positions: In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-101 of SEQ ID NO:66.In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 25-130 of SEQ ID NO: 66. In some embodiments, heteromultimers of the disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 36-101 of SEQ ID NO:66. In some embodiments, the heteromultimers of the present disclosure comprise at least one ALK5 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 36-130 of SEQ ID NO:66.

[0110] In certain aspects, the present disclosure relates to heteromultimers comprising ALK7 polypeptide.As used herein, the term "ALK7" refers to the family of activin receptor-like kinase-7 proteins from any organism and the mutants derived from such ALK7 proteins by mutagenesis or other modifications.In this specification, reference to ALK7 is understood to refer to any one of the currently identified forms.ALK7 family members are generally transmembrane proteins that are composed of a ligand-binding extracellular domain that includes a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain that has predicted serine / threonine kinase activity.

[0111] The term "ALK7 polypeptide" includes any naturally occurring polypeptide of the ALK7 family members as well as any variant thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retains useful activity. The amino acid numbering for all ALK7-related polypeptides described herein is based on the numbering of the human ALK7 precursor protein sequence (SEQ ID NO: 70, NCBI reference sequence NP_660302.2), unless specifically stated otherwise. In SEQ ID NO: 70, the signal peptide is indicated by a single underline and the extracellular domain is indicated by bold. The nucleic acid sequence encoding the human ALK7 isoform 1 precursor protein is shown in SEQ ID NO: 71, which is as per nucleotides 244-1722 of the Genbank reference sequence NM_145259.2.

[0112] The processed extracellular ALK7 isoform 1 polypeptide sequence is represented in SEQ ID NO: 72, which can be encoded by the nucleic acid sequence of SEQ ID NO: 73. The amino acid sequence of an alternative isoform of human ALK7, isoform 2 (NCBI Reference Sequence NP_001104501.1), is represented in SEQ ID NO: 74, with the extracellular domain indicated in bold. The nucleic acid sequence of the processed ALK7 polypeptide (isoform 2) is represented in SEQ ID NO: 75, which corresponds to nucleotides 279-1607 of NCBI Reference Sequence NP_001111031.1. The amino acid sequence of the extracellular ALK7 polypeptide (isoform 2) is represented in SEQ ID NO: 76, which can be encoded by the nucleic acid sequence of SEQ ID NO: 77. The amino acid sequence of an alternative human ALK7 precursor protein, isoform 3 (NCBI Reference Sequence NP_001104502.1), is represented in SEQ ID NO: 78, with the signal peptide indicated by single underlining. The nucleic acid sequence encoding the unprocessed ALK7 polypeptide precursor protein (isoform 3) is represented in SEQ ID NO: 79, which corresponds to nucleotides 244-1482 of the NCBI Reference Sequence NM_001111032.1. The signal sequence is indicated by a solid underline. The amino acid sequence of the processed ALK7 polypeptide (isoform 3) is represented in SEQ ID NO: 80. 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: 80 are predicted as follows: The nucleic acid sequence of the processed ALK7 polypeptide (isoform 3) is represented in SEQ ID NO: 81. The amino acid sequence of the alternative human ALK7 precursor protein, isoform 4 (NCBI Reference Sequence NP_001104503.1), is represented in SEQ ID NO: 82, with the signal peptide indicated by a single underline. The nucleic acid sequence encoding the unprocessed ALK7 polypeptide precursor protein (isoform 4) is represented in SEQ ID NO: 83, which corresponds to nucleotides 244 to 1244 of the NCBI reference sequence NM_001111033.1.The signal sequence is indicated by a solid underline. The amino acid sequence of the processed ALK7 polypeptide (isoform 4) is represented in SEQ ID NO: 84. Like ALK7 isoform 3, isoform 4 lacks the transmembrane domain and is therefore proposed to be soluble in its entirety (Roberts et al., 2003, Biol Reprod 68:1719-1726). The nucleic acid sequence of the processed ALK7 polypeptide (isoform 4) is represented in SEQ ID NO: 85.

[0113] Based on the signal sequence of full-length ALK7 (isoform 1) in rat (see NCBI reference sequence NP_620790.1) and the high degree of sequence identity between human ALK7 and rat ALK7, the processed form of human ALK7 isoform 1 is predicted to be represented by sequence number 86.

[0114] Active mutants of processed ALK7 isoform 1 are predicted, in which SEQ ID NO:72 is truncated by 1, 2, 3, 4, 5, 6, or 7 amino acids at the N-terminus, and SEQ ID NO:86 is truncated by 1 or 2 amino acids at the N-terminus. In agreement with SEQ ID NO:86, it is further predicted that leucine is the N-terminal amino acid in the processed forms of human ALK7 isoform 3 (SEQ ID NO:80) and human ALK7 isoform 4 (SEQ ID NO:84). In certain embodiments, the present disclosure relates to heteromultimers comprising at least one ALK7 polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the ALK7 polypeptides for use according to the disclosed invention (e.g., heteromultimers comprising ALK7 polypeptides and uses thereof) are soluble (e.g., the extracellular domain of ALK7). In other preferred embodiments, the ALK7 polypeptides for use according to the disclosed invention bind to one or more TGF-β superfamily ligands and / or inhibit (antagonize) their activity (e.g., Smad signaling). In some embodiments, the heteromultimers of the present disclosure comprise 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: 70, 72, 74, 76, 78, 80, 82, 84, or 86. In some embodiments, heteromultimers of the disclosure comprise at least one ALK7 polypeptide that is at least 70%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide beginning at any one of amino acids 21-28 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, or 28) of SEQ ID NO:70 and ending 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, or 113) of SEQ ID NO:70.In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 21-92 of SEQ ID NO: 70. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 21-113 of SEQ ID NO: 70. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 28-92 of SEQ ID NO: 70. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 28-113 of SEQ ID NO: 70. In some embodiments, heteromultimers of the disclosure comprise 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 a polypeptide starting at any one of amino acids 1-13 (e.g., amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) of SEQ ID NO:74 and ending at any one of amino acids 42-63 (e.g., amino acid residues 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63) of SEQ ID NO:74.In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 1-42 of SEQ ID NO: 74. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 1-63 of SEQ ID NO: 74. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 13-42 of SEQ ID NO: 74. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 13-63 of SEQ ID NO: 74. In some embodiments, heteromultimers of the disclosure comprise 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 a polypeptide beginning at any one of amino acids 21-28 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, or 28) of SEQ ID NO:78 and ending at any one of amino acids 411-413 (e.g., amino acid residues 411, 412, or 413) of SEQ ID NO:78. In some embodiments, the heteromultimers of the present disclosure comprise 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 amino acids 21-411 of SEQ ID NO:78.In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 21-413 of SEQ ID NO: 78. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 28-411 of SEQ ID NO: 78. In some embodiments, the heteromultimers of the present disclosure comprise 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 amino acids 28 to 413 of SEQ ID NO:78. In some embodiments, heteromultimers of the disclosure comprise 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 a polypeptide beginning at any one of amino acids 21-28 (e.g., amino acid residues 21, 22, 23, 24, 25, 26, 27, or 28) of SEQ ID NO:82 and ending at any one of amino acids 334-336 (e.g., amino acid residues 334, 335, or 336) of SEQ ID NO:82. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 21-334 of SEQ ID NO: 82. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 21-336 of SEQ ID NO:82.In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 28-334 of SEQ ID NO: 82. In some embodiments, heteromultimers of the disclosure comprise 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 amino acids 28-336 of SEQ ID NO:82.

[0115] The term "Crypto-1 polypeptide" includes polypeptides including any naturally occurring Crypto-1 protein (encoded by TDGF1 or one of its non-human orthologues) as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all Crypto-1 polypeptides described herein is based on the numbering of the human Crypto-1 precursor protein sequence (SEQ ID NO: 87, NCBI Reference Sequence NP_003203.1) unless specifically stated otherwise. The signal peptide is indicated by a single underline. The nucleic acid sequence encoding the unprocessed human Crypto-1 isoform 1 precursor protein is shown in SEQ ID NO: 88, which corresponds to nucleotides 385-948 of NCBI Reference Sequence NM_003212.3. The signal sequence is underlined. The processed Crypto-1 isoform 1 polypeptide sequence is represented in SEQ ID NO: 89, which can be encoded by the nucleic acid sequence of SEQ ID NO: 90. The human Crypto-1 isoform 2 protein sequence (NCBI Reference Sequence NP_001167607.1) is represented in SEQ ID NO: 91, which can be encoded by the nucleic acid sequence of SEQ ID NO: 92, which corresponds to nucleotides 43 to 558 of NCBI Reference Sequence NM_001174136.1. The processed extracellular Crypto-1 polypeptide sequence (isoform 2) is represented in SEQ ID NO: 93, which can be encoded by the nucleic acid sequence of SEQ ID NO: 94.

[0116] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the Crypto-1 polypeptide (e.g., heteromultimers comprising Crypto-1 polypeptides and their uses) for use according to the disclosed invention is soluble (e.g., the extracellular domain of Crypto-1). In other preferred embodiments, the Crypto-1 polypeptide for use according to the disclosed invention binds to and / or inhibits (antagonizes) the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, the heteromultimers of the present disclosure comprise at least one Crypto-1 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: 87, 89, 91, or 93. In some embodiments, the heteromultimer of the disclosure begins at any one of amino acids 31-82 of SEQ ID NO: 87 (e.g., amino acid residues 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, or 82); and at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide terminating at any one of amino acids 172 to 188 of SEQ ID NO: 87 (e.g., amino acid residues 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188).In some embodiments, heteromultimers of the present disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 31-188 of SEQ ID NO: 87. In some embodiments, heteromultimers of the present disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 63-172 of SEQ ID NO: 87. In some embodiments, heteromultimers of the present disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 82-172 of SEQ ID NO: 87. In some embodiments, heteromultimers of the present disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 82-188 of SEQ ID NO: 87. In some embodiments, heteromultimers of the present disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 31-172 of SEQ ID NO: 87. In some embodiments, heteromultimers of the present disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 63-188 of SEQ ID NO: 87.In some embodiments, the heteromultimer of the disclosure begins at any one of amino acids 15-66 of SEQ ID NO:91 (e.g., amino acid residues 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66); and at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide terminating at any one of amino acids 156 to 172 of SEQ ID NO: 91 (e.g., amino acid residues 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or 172). In some embodiments, heteromultimers of the disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 15-172 of SEQ ID NO: 91. In some embodiments, heteromultimers of the disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 47-172 of SEQ ID NO: 91. In some embodiments, heteromultimers of the present disclosure comprise at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 47 to 156 of SEQ ID NO:91.In some embodiments, heteromultimers of the disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 66-165 of SEQ ID NO: 91. In some embodiments, heteromultimers of the disclosure include at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 15-156 of SEQ ID NO: 91. In some embodiments, heteromultimers of the present disclosure comprise at least one Crypto-1 polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 66 to 172 of SEQ ID NO:91.

[0117] The term "cryptic polypeptide" includes polypeptides including any naturally occurring cryptic protein (encoded by CFC1 or one of its non-human orthologues) as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all cryptic polypeptides described herein is based on the numbering of the human cryptic isoform 1 precursor protein sequence (SEQ ID NO: 95, NCBI Reference Sequence NP_115934.1) unless specifically stated otherwise. The signal peptide is indicated by a single underline. The nucleic acid sequence encoding the unprocessed human cryptic isoform 1 precursor protein is shown in SEQ ID NO: 96, which corresponds to nucleotides 289-957 of NCBI Reference Sequence NM_032545.3. The signal sequence is underlined. The processed cryptic isoform 1 polypeptide sequence is depicted in SEQ ID NO: 97, which can be encoded by SEQ ID NO: 98. The human cryptic isoform 2 precursor protein (NCBI Reference Sequence NP_001257349.1) is represented in SEQ ID NO: 99. The signal peptide is indicated by a single underline. The nucleic acid sequence encoding the unprocessed human cryptic isoform 2 precursor protein is represented in SEQ ID NO: 100 and corresponds to nucleotides 289 to 861 of NCBI Reference Sequence NM_001270420.1. The signal sequence is underlined. The processed cryptic isoform 2 polypeptide sequence is represented in SEQ ID NO: 101, which can be encoded by the nucleic acid sequence of SEQ ID NO: 102. The human cryptic isoform 3 precursor protein sequence (NCBI Reference Sequence NP_001257350.1) is represented in SEQ ID NO: 103. The signal peptide is indicated by a single underline. The nucleic acid sequence encoding the unprocessed human cryptic isoform 3 precursor protein is represented in SEQ ID NO: 104 and corresponds to nucleotides 289 to 732 of the NCBI reference sequence NM_001270421.1. The signal sequence is underlined.The processed cryptic isoform 3 polypeptide sequence is depicted in SEQ ID NO:105, which can be encoded by the nucleic acid sequence of SEQ ID NO:106.

[0118] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the cryptic polypeptides for use according to the disclosed invention (e.g., heteromultimers comprising cryptic polypeptides and uses thereof) are soluble (e.g., the extracellular domain of cryptics). In other preferred embodiments, the cryptic polypeptides for use according to the disclosed invention bind to and / or antagonize the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, the heteromultimers of the present disclosure comprise at least one cryptic 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: 95, 97, 99, 101, 103, or 105.In some embodiments, the heteromultimers of the disclosure comprise amino acids 26-90 of SEQ ID NO:95 (e.g., amino acid residues 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90) and starting at any one of amino acids 157 to 233 of SEQ ID NO: 95 (e.g., amino acid residues 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 3 3, 174, 175, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 126, 217, 218, 219, 220 , 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, or 233). In some embodiments, the heteromultimers of the present disclosure include at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-233 of SEQ ID NO:95.In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-157 of SEQ ID NO: 95. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 90-157 of SEQ ID NO: 95. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-169 of SEQ ID NO: 95. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 90-169 of SEQ ID NO: 95. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 90-233 of SEQ ID NO: 95. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-82 of SEQ ID NO:95.In some embodiments, the heteromultimers of the disclosure begin at any one of amino acids 26-30 (e.g., amino acid residues 26, 27, 28, 29, or 30) of SEQ ID NO:99 and include any one of amino acids 82-191 (e.g., amino acid residues 82, 83, 84, 85, 86, 57, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, In some embodiments, the heteromultimers of the present disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of SEQ ID NO: 183, 184, 185, 186, 187, 188, 189, 190, or 191. In some embodiments, the heteromultimers of the present disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-82 of SEQ ID NO: 99. In some embodiments, the heteromultimers of the present disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-191 of SEQ ID NO:99.In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-82 of SEQ ID NO: 99. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-191 of SEQ ID NO:99. In some embodiments, the heteromultimer of the present disclosure begins at any one of amino acids 26-30 (e.g., amino acid residues 26, 27, 28, 29, or 30) of SEQ ID NO: 103 and includes any one of amino acids 82-148 (e.g., amino acid residues 82, 83, 84, 85, 86, 57, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or 230) of SEQ ID NO: 103 and includes any one of amino acids 82-148 (e.g., amino acid residues 82, 83, 84, 85, 86, 57, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, or 148). In some embodiments, the heteromultimers of the present disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-148 of SEQ ID NO:103.In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-82 of SEQ ID NO: 103. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-148 of SEQ ID NO: 103. In some embodiments, the heteromultimer of the present disclosure comprises at least one cryptic polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30 to 82 of SEQ ID NO:103.

[0119] The term "cryptic family protein 1B polypeptide" (cryptic 1B) includes any naturally occurring cryptic family protein 1B protein (encoded by CFC1B or one of its non-human orthologues) as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. The amino acid numbering for all cryptic family protein 1B related polypeptides described herein is based on the numbering of the human cryptic family protein 1B precursor protein sequence (SEQ ID NO: 107, NCBI Reference Sequence NP_001072998.1, signal peptide indicated by single underline), unless specifically stated otherwise. The nucleic acid sequence encoding the unprocessed cryptic family protein 1B precursor protein is shown in SEQ ID NO: 108, which corresponds to nucleotides 392 to 1060 of NCBI Reference Sequence NM_001079530.1. The signal sequence is underlined. The processed cryptic family protein 1B polypeptide sequence is represented in SEQ ID NO:109, which can be encoded by the nucleic acid sequence of SEQ ID NO:110.

[0120] In certain embodiments, the present disclosure relates to heteromultimers comprising at least one cryptic family protein 1B polypeptide (including fragments, functional variants, and modified forms thereof). Preferably, the cryptic family protein 1B polypeptide for use according to the disclosed invention (e.g., heteromultimers comprising cryptic family protein 1B polypeptides and uses thereof) is soluble (e.g., the extracellular domain of cryptic family protein 1B). In other preferred embodiments, the cryptic family protein 1B polypeptide for use according to the disclosed invention binds to and / or antagonizes the activity (e.g., Smad signaling) of one or more TGF-β superfamily ligands. In some embodiments, the heteromultimer of the present disclosure comprises at least one cryptic family protein 1B 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: 107 or 109.In some embodiments, the heteromultimer of the disclosure begins at any one of amino acids 26-30 (e.g., amino acid residues 26, 27, 28, 29, or 30) of SEQ ID NO: 107 and includes any one of amino acids 82-223 (e.g., amino acid residues 82, 83, 84, 85, 86, 57, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 401, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 07, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 1 57, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 2 The present invention relates to a cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide that terminates at any one of the following positions: 09, 210, 211, 212, 213, 214, 215, 126, 217, 218, 219, 220, 221, 222, or 223. In some embodiments, the heteromultimer of the present disclosure comprises at least one cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26 to 223 of SEQ ID NO:107.In some embodiments, heteromultimers of the disclosure comprise at least one cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-82 of SEQ ID NO: 107. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-82 of SEQ ID NO: 107. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-223 of SEQ ID NO: 107. In some embodiments, heteromultimers of the disclosure comprise at least one cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 26-169 of SEQ ID NO: 107. In some embodiments, the heteromultimer of the present disclosure comprises at least one cryptic family protein 1B polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30 to 169 of SEQ ID NO:107.

[0121] In certain aspects, the present disclosure relates to homomultimers comprising at least two ALK4 polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the ALK4 polypeptides described herein). In certain preferred embodiments, the ALK4 homomultimers of the present disclosure are homodimers.

[0122] In certain aspects, the present disclosure relates to homomultimers comprising at least two ALK5 polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the ALK5 polypeptides described herein). In certain preferred embodiments, the ALK5 homomultimers of the present disclosure are homodimers.

[0123] In certain aspects, the present disclosure relates to homomultimers comprising at least two ALK7 polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the ALK7 polypeptides described herein). In certain preferred embodiments, the ALK7 homomultimers of the present disclosure are homodimers.

[0124] In certain aspects, the present disclosure relates to homomultimers comprising at least two ActRIIA polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the ActRIIA polypeptides described herein). In certain preferred embodiments, the ActRIIA homomultimers of the present disclosure are homodimers.

[0125] In certain aspects, the present disclosure relates to homomultimers comprising at least two ActRIIB polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the ActRIIB polypeptides described herein). In certain preferred embodiments, the ActRIIB homomultimers of the present disclosure are homodimers.

[0126] In certain aspects, the present disclosure relates to homomultimers comprising at least two Crypto-1 polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the Crypto-1 polypeptides described herein). In certain preferred embodiments, the Crypto-1 homomultimers of the present disclosure are homodimers.

[0127] In certain aspects, the present disclosure relates to homomultimers comprising at least two cryptic polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the cryptic polypeptides described herein). In certain preferred embodiments, the cryptic homomultimers of the present disclosure are homodimers.

[0128] In certain aspects, the present disclosure relates to homomultimers comprising at least two cryptic 1B polypeptides, including fragments, functional variants, and modified forms thereof (e.g., any of the cryptic 1B polypeptides described herein). In certain preferred embodiments, the cryptic 1B homomultimers of the present disclosure are homodimers.

[0129] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK1:ActRIIB heteromultimer complex of the present disclosure is a heterodimer.

[0130] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK4:ActRIIB heteromultimer complex of the present disclosure is a heterodimer.

[0131] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK5:ActRIIB heteromultimer complex of the present disclosure is a heterodimer.

[0132] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK7:ActRIIB heteromultimer complex of the present disclosure is a heterodimer.

[0133] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK1:ActRIIA heteromultimer complex of the present disclosure is a heterodimer.

[0134] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK4:ActRIIA heteromultimer complex of the present disclosure is a heterodimer.

[0135] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK5:ActRIIA heteromultimer complex of the present disclosure is a heterodimer.

[0136] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK7:ActRIIA heteromultimer complex of the present disclosure is a heterodimer.

[0137] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK4:BMPRII heteromultimer complex of the present disclosure is a heterodimer.

[0138] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK5:BMPRII heteromultimer complex of the present disclosure is a heterodimer.

[0139] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK7:BMPRII heteromultimer complex of the present disclosure is a heterodimer.

[0140] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein).In certain preferred embodiments, the ALK4:MISRII heteromultimer complex of the present disclosure is a heterodimer.

[0141] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein).In certain preferred embodiments, the ALK5:MISRII heteromultimer complex of the present disclosure is a heterodimer.

[0142] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein).In certain preferred embodiments, the ALK7:MISRII heteromultimer complex of the present disclosure is a heterodimer.

[0143] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK1:ALK4 heteromultimers of the present disclosure are heterodimers.

[0144] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK1:ALK5 heteromultimers of the present disclosure are heterodimers.

[0145] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK1:ALK7 heteromultimers of the present disclosure are heterodimers.

[0146] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK4:ALK5 heteromultimers of the present disclosure are heterodimers.

[0147] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK4:ALK7 heteromultimers of the present disclosure are heterodimers.

[0148] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ALK5:ALK7 heteromultimers of the present disclosure are heterodimers.

[0149] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ActRIIA:ActRIIB heteromultimers of the present disclosure are heterodimers.

[0150] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ActRIIA:BMPRII heteromultimers of the present disclosure are heterodimers.

[0151] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ActRIIA:MISRII heteromultimers of the present disclosure are heterodimers.

[0152] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ActRIIB:BMPRII heteromultimers of the present disclosure are heterodimers.

[0153] In certain aspects, the present disclosure relates to heteromultimers comprising at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the ActRIIB:MISRII heteromultimer of the present disclosure is a heterodimer.

[0154] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:ALK1 heteromultimers of the present disclosure are heterodimers.

[0155] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:ALK4 heteromultimer of the present disclosure is a heterodimer.

[0156] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:ALK5 heteromultimer of the present disclosure is a heterodimer.

[0157] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:ALK7 heteromultimer of the present disclosure is a heterodimer.

[0158] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:ALK1 heteromultimers of the present disclosure are heterodimers.

[0159] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:ALK4 heteromultimers of the present disclosure are heterodimers.

[0160] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:ALK5 heteromultimers of the present disclosure are heterodimers.

[0161] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:ALK7 heteromultimers of the present disclosure are heterodimers.

[0162] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:ALK1 heteromultimers of the present disclosure are heterodimers.

[0163] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK4 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:ALK4 heteromultimers of the present disclosure are heterodimers.

[0164] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK5 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:ALK5 heteromultimers of the present disclosure are heterodimers.

[0165] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ALK7 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:ALK7 heteromultimers of the present disclosure are heterodimers.

[0166] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:ActRIIA heteromultimers of the present disclosure are heterodimers.

[0167] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:ActRIIB heteromultimer of the present disclosure is a heterodimer.

[0168] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:BMPRII heteromultimer of the present disclosure is a heterodimer.

[0169] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:MISRII heteromultimer of the present disclosure is a heterodimer.

[0170] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:ActRIIA heteromultimers of the present disclosure are heterodimers.

[0171] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:ActRIIB heteromultimers of the present disclosure are heterodimers.

[0172] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:BMPRII heteromultimers of the present disclosure are heterodimers.

[0173] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:MISRII heteromultimers of the present disclosure are heterodimers.

[0174] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIA polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:ActRIIA heteromultimers of the present disclosure are heterodimers.

[0175] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one ActRIIB polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:ActRIIB heteromultimers of the present disclosure are heterodimers.

[0176] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one BMPRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:BMPRII heteromultimers of the present disclosure are heterodimers.

[0177] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one MISRII polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic 1B:MISRII heteromultimers of the present disclosure are heterodimers.

[0178] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one Cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:cryptic polypeptide heteromultimers of the present disclosure are heterodimers.

[0179] In certain aspects, the present disclosure relates to heteromultimers comprising at least one Crypto-1 polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one Cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the Crypto-1:Cryptic 1B heteromultimer complex of the present disclosure is a heterodimer.

[0180] In certain aspects, the present disclosure relates to heteromultimers comprising at least one cryptic polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein), and at least one cryptic 1B polypeptide, including fragments, functional variants, and modified forms thereof (e.g., any of those described herein). In certain preferred embodiments, the cryptic polypeptide:cryptic 1B heteromultimer is soluble. In some embodiments, the cryptic polypeptide:cryptic 1B heteromultimer of the present disclosure is a heterodimer.

[0181] In some embodiments, the present disclosure contemplates generating functional variants by modifying the structure of TGF-β superfamily type I receptor polypeptides (e.g., ALK1, ALK4, ALK5, and ALK7), TGF-β superfamily type II receptor polypeptides (e.g., ActRIIA, ActRIIB, BMPRII, and MISRII), and / or TGF-β superfamily coreceptors (e.g., Crypto-1, Cryptic, and Cryptic 1B) for purposes such as enhancing therapeutic efficacy or stability (e.g., shelf life and resistance to proteolysis in vivo). Variants can be generated by amino acid substitution, deletion, addition, or combinations thereof. For example, it is reasonable to expect that single substitutions of leucine to isoleucine or valine, aspartic acid to glutamic acid, threonine to serine, or similar substitutions of one amino acid to a structurally related amino acid (e.g., conservative mutations) will not have a significant effect on the biological activity of the resulting molecule. Conservative substitutions are made within a family of amino acids whose side chains are related. Whether a change in the amino acid sequence of a polypeptide of the present disclosure results in a functional homolog can be readily determined by assessing the ability of the mutant polypeptide to elicit a response in a cell in a manner similar to the wild-type polypeptide or to bind to one or more TGF-β superfamily ligands (including, for example, GDF3, GDF5, GDF1, GDF8, GDF11, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, activin BE, nodal).

[0182] In certain embodiments, the present disclosure contemplates specific mutations of the disclosed TGF-β superfamily type I receptor polypeptides (e.g., ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7), TGF-β superfamily type II receptor polypeptides (e.g., ActRIIA, ActRIIB, BMPRII, and MISRII), and / or TGF-β superfamily coreceptor polypeptides (e.g., endoglin, betaglycan, crypt-1, cryptic, cryptic 1B, CRIM1, CRIM2, BAMBI, BMPER, RGM-A, RGM-B, and hemojuvelin) that alter the glycosylation of the polypeptide. Such mutations can be selected to introduce or remove one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally contain a tripeptide sequence of asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid), which is specifically recognized by the glycosylation enzymes of the appropriate cell. Alterations can also be made by the addition of, or substitution by, one or more serine or threonine residues (O-linked glycosylation sites) to the sequence of the polypeptide. Various amino acid substitutions or deletions at either or both of the first or third amino acid positions (and / or deletion of an amino acid at the second position) of the glycosylation recognition site result in non-glycosylation of the altered 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 method used, sugars are linked to (a) arginines and histidines; (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 the polypeptide can be accomplished chemically and / or enzymatically.Chemical deglycosylation can include, for example, exposure of the 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 the polypeptide can be achieved through the use of various endoglycosidases and exoglycosidases, such as those described by Thotakura et al. [Meth. Enzymol. (1987) 138:350]. Mammalian cells, yeast cells, insect cells, and plant cells can all introduce different glycosylation patterns, which can be influenced by the amino acid sequence of the peptide, so the sequence of the polypeptide can be adjusted accordingly depending on the type of expression system used. Generally, the heteromultimers of the present disclosure for use in humans can be expressed in mammalian cell lines (such as HEK293 or CHO cell lines) that provide proper glycosylation, although it is expected that other mammalian expression cell lines will be useful as well.

[0183] The present disclosure further contemplates methods for generating mutants, particularly combinatorial mutant sets of the disclosed TGF-β superfamily type I receptor polypeptides (e.g., ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7), TGF-β superfamily type II receptor polypeptides (e.g., ActRIIA, ActRIIB, BMPRII, and MISRII), and / or TGF-β superfamily coreceptor polypeptides (e.g., endoglin, betaglycan, crypt-1, cryptic, cryptic 1B, CRIM1, CRIM2, BAMBI, BMPER, RGM-A, RGM-B, and hemojuvelin), as well as truncation mutants. Pools of combinatorial mutants are particularly useful for identifying functionally active (e.g., ligand-binding) TGF-β superfamily type I receptors, TGF-β superfamily type II receptors, and / or TGF-β superfamily coreceptor sequences. The purpose of screening such combinatorial libraries can be, for example, to generate polypeptide variants with altered properties, such as altered pharmacokinetics or altered ligand binding. A variety of screening assays are provided below that can be used to evaluate the variants.For example, the TGF-β co-receptor mutants may be TGF-β superfamily ligands (e.g., BMP2, BMP2 / 7, BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP13, GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF15 / MIC1, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, The antibodies can be screened for their ability to bind to activin BC, activin BE, Nodal, glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and lefty, inhibit the binding of a TGF-β superfamily ligand to a TGF-β superfamily co-receptor, and / or disrupt signal transduction caused by a TGF-β superfamily ligand.

[0184] The activity of the TGF-β superfamily receptor polypeptides (including heteromultimers and homomultimers) of the present disclosure can also be tested, for example, in cell-based or in vivo assays. For example, the effect of the TGF-β superfamily receptor polypeptides on the expression of genes or activity of proteins involved in muscle production in muscle cells can be evaluated. This can be done, if necessary, by incorporation of one or more recombinant TGF-β superfamily ligand proteins (e.g., BMP2, BMP2 / 7, BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP13, GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF15 / MIC1, activin A, activin B, activin C, activin E, activin AB ... , activin AC, activin AE, activin BC, activin BE, nodal, glial cell line derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and lefty), and the cells can be transfected to produce TGF-β superfamily receptor polypeptides and optionally TGF-β superfamily ligands. Similarly, the TGF-β superfamily receptor polypeptides of the present disclosure can be administered to mice or other animals, and one or more measurements, such as muscle formation and strength, can be assessed using art-recognized methods. Similarly, the activity of the heteromultimers, or variants thereof, can be tested in osteoblasts, adipocytes, and / or neuronal cells for any effect on the growth of these cells, for example, by assays described herein and known in the art. SMAD-responsive reporter genes can be used in such cell lines to monitor for effects on downstream signaling.

[0185] Combinatorially derived variants can be generated that have increased selectivity or generally increased potency compared to a reference TGF-β superfamily receptor polypeptide. Such variants can be used in gene therapy protocols when expressed from recombinant DNA constructs. Similarly, mutagenesis can result in variants that have dramatically different intracellular half-lives than the corresponding unmodified TGF-β superfamily receptor polypeptide. For example, modified proteins can be made more stable or less stable to proteolysis or other cellular processing that would destroy or otherwise inactivate the unmodified polypeptide. Such variants, and the genes that encode them, can be used to alter the polypeptide complex levels by altering the polypeptide half-life. For example, a shorter half-life can result in a more transient biological effect and, when part of an inducible expression system, can allow for tighter control of recombinant polypeptide complex levels in cells. In an Fc fusion protein, mutations can be made in the linker (if any) and / or the Fc portion to alter one or more activities of the TGF-β superfamily receptor polypeptide, including, for example, immunogenicity, half-life, and solubility.

[0186] Combinatorial libraries can be generated using degenerate libraries of genes that encode libraries of polypeptides each containing at least a portion of a potential TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that a degenerate set of potential TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor encoding nucleotide sequences can be expressed as individual polypeptides or as a set of larger fusion proteins (e.g., for phage display).

[0187] There are many ways that a library of potential homologues can be made from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automatic DNA synthesizer, and the synthetic gene can then be ligated into a suitable vector for expression. The synthesis of degenerate oligonucleotides is well known in the art. For example, see Narang, SA (1983) Tetrahedron 39:3;Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289;Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056;Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been used in the targeted evolution of other proteins. See, e.g., Scott et al., (1990) Science 249:386-390; Roberts et al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and U.S. Patent Nos. 5,223,409, 5,198,346, and 5,096,815.

[0188] Alternatively, other forms of mutagenesis can be used to generate combinatorial libraries. For example, heteromultimers of the present disclosure can be made and isolated from libraries by screening, for example, using the following methods: alanine scanning mutagenesis [e.g., Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085], linker scanning mutagenesis [see, e.g., 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], saturation mutagenesis [see, e.g., Meyers et al., (1986) Science 232:613]; PCR mutagenesis [see, e.g., Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or random mutagenesis, including chemical mutagenesis [see, e.g., Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol See Biol 7:32-34.Linker scanning mutagenesis is an attractive method for identifying truncated (bioactive) forms of TGF-β superfamily type I receptor, type II receptor, and / or co-receptor polypeptides, especially in combinatorial conditions.

[0189] A wide range of techniques are known in the art for screening the gene products of combinatorial libraries made by point mutation and truncation, and for screening cDNA libraries for gene products with specific properties.Such techniques will generally be applicable to the rapid screening of gene libraries made by combinatorial mutagenesis of heteromultimers of the present disclosure.The most widely used techniques for screening large gene libraries typically include cloning gene libraries into replicable expression vectors, transforming suitable cells with the resulting library of vectors, and expressing combinatorial genes under conditions where detection of desired activity facilitates the relatively easy isolation of the vectors encoding the genes whose products are detected. Preferred assays include TGF-β superfamily ligand (e.g., BMP2, BMP2 / 7, BMP3, BMP4, BMP4 / 7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5, GDF6 / BMP13, GDF7, GDF8, GDF9b / BMP15, GDF11 / BMP11, GDF15 / MIC1, activin A, activin B, activin C, activin E, activin AB, activin AC, activin AE, activin BC, activin BE, nodal, glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin, persephin, MIS, and lefty) binding assays and / or TGF-β superfamily ligand-mediated signaling assays.

[0190] In certain embodiments, the heteromultimeric TGF-β superfamily receptor polypeptides of the present disclosure (including their heteromultimers and homomultimers) can further include post-translational modifications in addition to any that naturally occur in TGF-β superfamily type I receptor, type II receptor, or co-receptor polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, TGF-β superfamily receptor polypeptides can include non-amino acid components such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid components on the functionality of TGF-β superfamily receptor polypeptides can be tested as described herein for other TGF-β superfamily receptor polypeptide variants. When the polypeptides of the present disclosure are produced in cells by cleaving the nascent form of the polypeptide, post-translational processing can also be important for the proper folding and / or function of the protein. Various cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific and characteristic intracellular mechanisms for such post-translational activity and can be selected to ensure proper modification and processing of TGF-β superfamily type I receptor, type II receptor, and / or co-receptor polypeptides and heteromultimers containing same.

[0191] In some embodiments, the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide further comprises one or more heterologous moieties (e.g., a polypeptide comprising a TGF-β superfamily type I receptor polypeptide domain and a second polypeptide domain heterologous to the TGF-β superfamily type I receptor polypeptide domain), thereby conferring desired properties. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. For the purpose of affinity purification, relevant matrices for affinity chromatography are used, such as glutathione-conjugated, amylase-conjugated, and nickel- or cobalt-conjugated resins. Many such matrices are available in the Pharmacia GST purification system and (HIS 6 ) Useful QIAexpress fusion partners TMAs another example, the fusion domain may be selected to facilitate detection of the polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) as well as epitope tags (usually short peptide sequences for which specific antibodies are available). Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain contains a protease cleavage site, such as those for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein from the fusion protein. The released proteins are then subjected to subsequent chromatographic separation. It can be isolated from the fusion domain.

[0192] In some embodiments, the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide of the present disclosure comprises one or more modifications that can stabilize the polypeptide. For example, such modifications enhance the in vitro half-life of the polypeptide, enhance the circulating half-life of the polypeptide, and / or reduce the proteolysis of the polypeptide. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising a type I receptor polypeptide, a type II receptor polypeptide, or a co-receptor polypeptide domain and a stabilizer domain), modifications of glycosylation sites (including, for example, adding a glycosylation site to a polypeptide of the present disclosure), and modifications of carbohydrate moieties (including, for example, removing a carbohydrate moiety from a polypeptide of the present disclosure). As used herein, "stabilizer domain" does not mean only the fusion domain (e.g., immunoglobulin Fc domain) as in the case of a fusion protein, but also includes non-proteinaceous modifications such as carbohydrate moieties, or non-proteinaceous moieties such as polyethylene glycol.

[0193] It is understood that the different components of the fusion protein (e.g., immunoglobulin Fc fusion protein) can be arranged in any manner consistent with the desired functionality. For example, the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide domain can be arranged C-terminal to the heterologous domain, or the heterologous domain can be arranged C-terminal to the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide domain. The TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor domain and the heterologous domain do not need to be adjacent in the fusion protein, and additional domains or amino acid sequences can be included at the C-terminus or N-terminus of either domain, or between the domains.

[0194] For example, a TGF-β superfamily type I receptor, type II receptor, or co-receptor fusion protein can comprise an amino acid sequence represented by the formula ABC. The B portion corresponds to a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide domain. The A and C portions can independently be zero, one, or more amino acids, and when both the A and C portions are present, are heterologous to B. The A and / or C portions can be linked to the B portion via a linker sequence. The linker can be glycine-rich (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or rich in glycine and proline residues, and can include, for example, single sequences of threonine / serine and glycine or repeating sequences of threonine / serine and / or glycine (e.g., GGG (SEQ ID NO: 223), GGGG (SEQ ID NO: 222), TGGGG (SEQ ID NO: 219), SGGGG (SEQ ID NO: 220), TGGG (SEQ ID NO: 217), GGGGS (SEQ ID NO: 221), or SGGG (SEQ ID NO: 218)), singlets, or repeats. In certain embodiments, the TGF-β superfamily type I receptor, type II receptor, or co-receptor fusion protein comprises an amino acid sequence represented by the formula ABC, where A is a leader (signal) sequence, B is a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide domain, and C is a polypeptide moiety that enhances one or more of in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. In certain embodiments, the TGF-β superfamily type I receptor, type II receptor, or co-receptor fusion protein comprises an amino acid sequence represented by the formula ABC, where A is a TPA leader sequence, B is a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide domain, and C is an immunoglobulin Fc domain.

[0195] As a specific example, the present disclosure provides a fusion protein comprising a TGF-β superfamily type I receptor, type II receptor, or co-receptor polypeptide fused to a polypeptide comprising an immunoglobulin constant domain, such as the CH1, CH2, or CH3 domain of an immunoglobulin Fc domain. Fc domains derived from human IgG1, IgG2, IgG3, and IgG4 are provided herein. Other mutations that reduce either CDC or ADCC activity are known, and collectively, any of these variants are included in the present disclosure and can be used as beneficial components of the heteromultimers of the present disclosure. Optionally, the IgG1 Fc domain of SEQ ID NO: 135 has one or more mutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered according to the corresponding full-length IgG1). In certain cases, mutant Fc domains with one or more of these mutations (e.g., an Asp-265 mutation) have reduced binding ability to Fcγ receptors compared to wild-type Fc domains. In other cases, mutant Fc domains having one or more of these mutations (e.g., an Asn-434 mutation) have increased binding ability to MHC class I-associated Fc receptors (FcRN) compared to wild-type Fc domains.

[0196] An example of a native amino acid sequence that can be used for the Fc portion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 135). The dotted underline indicates the hinge region, and the solid underline indicates the position with a naturally occurring variant. In part, the present disclosure provides a polypeptide comprising an amino acid sequence having 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 135. Naturally occurring variants in G1Fc would include E134D and M136L according to the numbering system used in SEQ ID NO: 135 (see Uniprot P01857).

[0197] An example of a native amino acid sequence that can be used for the Fc portion of human IgG2 (G2Fc) is shown below (SEQ ID NO:136). The dotted underline indicates the hinge region and the double underline indicates positions in the sequence where there is a database discrepancy (according to UniProt P01859). In part, the disclosure provides polypeptides comprising an amino acid sequence having 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:136.

[0198] Two examples of amino acid sequences that can be used for the Fc portion of human IgG3 (G3Fc) are shown below. The hinge region in G3Fc, which can be up to four times as in other Fc chains, contains three identical 15-residue segments preceded by a similar 17-residue segment. The first G3Fc sequence shown below (SEQ ID NO: 137) contains a short hinge region consisting of a single 15-residue segment, while the second G3Fc sequence (SEQ ID NO: 138) contains a full-length hinge region. In each case, the dotted underline indicates the hinge region and the solid underline indicates the position with a naturally occurring mutation according to UniProt P01859. In part, the disclosure provides polypeptides comprising an amino acid sequence having 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:137 or 138.

[0199] Naturally occurring variants in G3Fc (see, e.g., Uniprot P01860) include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del, F221Y (when converted to the numbering system used in SEQ ID NO: 137), and the present disclosure provides fusion proteins comprising a G3Fc domain that includes one or more of these variants. In addition, the human immunoglobulin IgG3 gene (IGHG3) shows a structural polymorphism characterized by different hinge lengths (see Uniprot P01859). Specifically, variant WIS lacks most of the V region and all of the CH1 region. This variant has an extra interchain disulfide bond at position 7 in addition to the 11 normally present in the hinge region. Variant ZUC lacks the step portion of the V region, all of the CH1 region, and part of the hinge. The variant OMMs can represent allelic forms or alternative gamma chain subclasses. The present disclosure provides additional fusion proteins comprising a G3Fc domain containing one or more of these variants.

[0200] An example of a native amino acid sequence that can be used for the Fc portion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 139). The dotted underline indicates the hinge region. In part, the disclosure provides polypeptides comprising an amino acid sequence having 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 139.

[0201] Various engineered mutations in the Fc domain are presented herein with respect to the G1Fc sequence (SEQ ID NO: 135), and similar mutations in G2Fc, G3Fc, and G4Fc can be derived from their alignment with G1Fc in Figure 4. Due to the uneven hinge lengths, analogous Fc positions based on the isotype alignment (Figure 4) have different amino acid numbers in SEQ ID NOs: 135, 136, 137, and 139. Hinge, C H 2, and C HA given amino acid position in a three-region immunoglobulin sequence (e.g., SEQ ID NO: 135, 136, 137, 138, or 139) is numbered according to the full-length IgG1 heavy chain constant domain (C H 1, hinge, C H 2, and C H It is also understood that in cases involving a sequence that includes a sequence that is a conserved region of the human G1Fc sequence (SEQ ID NO: 135), a sequence that is a conserved region of the human IgG1 heavy chain (Uniprot P01857), and a sequence that is a conserved region of the human IgG1 heavy chain (Uniprot P01857), the same positions will be identified by different numbers. H The correspondence between the three positions is as follows:

[0202] [Table 1]

[0203] In certain embodiments, a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide (e.g., those described herein) can covalently or non-covalently form a heteromultimer with at least one additional TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide. Numerous methods known in the art can be used to create heteromultimers. For example, a non-naturally occurring disulfide bond can be constructed by replacing a naturally occurring amino acid on a first polypeptide (e.g., a TGF-β superfamily type I polypeptide) with a free thiol-containing residue, such as cysteine, so that the free thiol interacts with another thiol-containing residue on a second polypeptide (e.g., a TGF-β superfamily type II polypeptide) to form a disulfide bond between the first and second polypeptides. Additional examples of interactions for promoting heteromultimer formation include, but are not limited to, ionic interactions such as those described in Kjaergaard et al., WO 2007147901; electrostatic steering effects such as those described in Kannan et al., U.S. Pat. No. 8,592,562; coiled-coil interactions such as those described in Christensen et al., U.S. Pat. App. Pub. No. 20120302737; leucine zippers such as those described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584; and helix-turn-helix motifs such as those described in Pack et al., (1993) Bio / Technology 11: 1271-1277. Linkage of the various segments can be achieved, for example, through covalent bonds such as chemical crosslinks, peptide linkers, disulfide bridges, or affinity interactions such as by biotin-avidin or leucine zipper technology.Preferably, the polypeptides disclosed herein form heterodimers, but also include higher order heteromultimers, such as, but not limited to, heterotrimers, heterotetramers, and further aryl structures (see, e.g., Figures 5 and 6).

[0204] In some embodiments, the TGF-β superfamily type I receptor, type II receptor, and / or co-receptor polypeptide of the present disclosure comprises at least one multimerization domain. As disclosed herein, the term "multimerization domain" refers to an amino acid or sequence of amino acids that promotes covalent or non-covalent interaction between at least a first polypeptide and at least a second polypeptide. The polypeptides disclosed herein can be covalently or non-covalently linked to the multimerization domain. In some embodiments, the multimerization domain promotes the interaction between a first polypeptide and a second polypeptide to promote heteromultimer formation (e.g., heterodimer formation), and optionally prevents or otherwise disfavors homomultimer formation (e.g., homodimer formation) to increase the yield of the desired heteromultimer (see, for example, Figures 5 and 6).

[0205] In certain aspects, the multimerization domain can comprise one member of an interaction pair. In some embodiments, the polypeptides of the present disclosure can form a protein complex comprising a first polypeptide covalently or non-covalently associated with a second polypeptide, where the first polypeptide comprises the amino acid sequence of a first TGF-β superfamily receptor polypeptide and the amino acid sequence of a first member of the interaction pair; and the second polypeptide comprises the amino acid sequence of a second TGF-β superfamily receptor polypeptide and the amino acid sequence of a second member of the interaction pair. Although functional embodiments can also utilize an interaction pair capable of forming a homodimeric complex, the interaction pair can be any two polypeptide sequences that interact to form a complex, particularly a heterodimeric complex. The interaction pair can be selected to confer an improved property / activity, such as an increased serum half-life, or to act as an adapter to which another moiety is linked to provide an improved property / activity. For example, a polyethylene glycol moiety can be linked to one or both members of the interaction pair to provide an improved property / activity, such as an improved serum half-life.

[0206] The first and second members of the interaction pair can be asymmetric pairs, which means that the members of the pair preferentially associate with each other, rather than self-associating. Thus, the first and second members of the asymmetric interaction pair can associate to form, for example, a heterodimeric complex (see, for example, Figures 5A and 5B). Alternatively, the interaction pair can be unguided, which means that the members of the pair can associate with each other or self-associate without substantial preference, that is, they can have the same or different amino acid sequences. Thus, the first and second members of the unguided interaction pair can associate to form a homodimeric complex or a heterodimeric complex. Optionally, the first member of the interaction pair (e.g., asymmetric or unguided interaction pair) is covalently associated with the second member of the interaction pair. Optionally, the first member of the interaction pair (e.g., asymmetric or unguided interaction pair) is non-covalently associated with the second member of the interaction pair.

[0207] A problem that arises in the large-scale production of asymmetric immunoglobulin-based proteins from a single cell line is known as the "chain association issue." As prominently presented in the production of bispecific antibodies, the chain association issue relates to the difficulty of efficiently producing the desired multi-chain protein from the multiple combinations that arise inherently when different heavy 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 pressing when two different heavy chains and two different light chains are produced in the same cell, resulting in a total of 16 possible chain combinations, some of which are identical, when only one is typically desired. Nevertheless, the same principle accounts for the reduced yield of the desired multi-chain fusion protein that incorporates only two (asymmetric) heavy chains.

[0208] Various methods are known in the art to increase the desired pairing of Fc-containing fusion polypeptide chains in a single cell line to produce acceptable yields of preferred asymmetric fusion proteins (see, for example, Klein et al (2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology 67(2A): 95-106). Methods for obtaining the desired pairing of Fc-containing chains include, but are not limited to, charge-based pairing (electrostatic induction), "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. Patent No. 5,932,448; WO 1993 / 011162; WO 2009 / 089004, and WO 2011 / 034605. As described herein, these methods can be used to generate heterodimers comprising two or more TGF-β superfamily receptor polypeptides. See Figures 5 and 6.

[0209] For example, one means by which interactions between specific polypeptides can be promoted is by genetically engineering protuberance-into-cavity (knob-into-holes) complementary regions (such as those described in Arathoon et al., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168). "Protuberances" can be constructed by replacing small amino acid side chains at the interface of a 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 a second polypeptide (e.g., a second interaction pair) by replacing the large amino acid side chains with relatively small ones (e.g., alanine or threonine). If a protrusion or cavity with suitable location and dimensions is present on the interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protrusion, respectively, into the adjacent interface.

[0210] At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged, while lysine, arginine, and histidine are positively charged. These charged residues can be used to promote heterodimer formation and simultaneously hinder homodimer formation. Attractive interactions occur between opposite charges, and repulsive interactions occur between similar charges. In part, the protein complexes disclosed herein use attractive interactions for heteromultimer formation (e.g., heterodimer formation) and, optionally, repulsive interactions to hinder homodimer formation (e.g., homodimer formation) by performing site-directed mutagenesis of charged surface residues.

[0211] For example, the IgG1 CH3 domain interface contains four unique pairs of charged residues involved in interdomain interactions: Asp356-Lys439', Glu357-Lys370', Lys392-Asp399', and Asp399-Lys409' (residue numbering in the second chain is indicated by ('). Note that the numbering system used herein to assign residues in the IgG1 CH3 domain is aligned with the EU numbering system of Kabat. Due to the two-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction will appear twice in the structure (e.g., Asp-399-Lys409' and Lys409-Asp399'). In the wild-type sequence, K409-D399' favors the formation of both heterodimers and homodimers. A single mutation that switches the charge polarity in the first strand (e.g., K409E; from positive to negative) results in an unfavorable interaction for the formation of first strand homodimers. The unfavorable interaction occurs due to repulsive interactions between like charges (negative and negative; K409E-D399' and D399-K409E'). A similar mutation that switches the charge polarity in the second strand (D399K'; from negative to positive) results in an unfavorable interaction for the formation of second strand homodimers (K409'-D399K' and D399K-K409'). However, at the same time, two mutations (K409E and D399K') result in a favorable interaction for heterodimer formation (K409E-D399K' and D399-K409').

[0212] The electrostatic steering effect on heterodimer formation and homodimer inhibition can be further enhanced by mutation of additional charged residues that may or may not pair with oppositely charged residues in the second strand, including, for example, Arg355 and Lys360. Table 2 below lists possible charge-altering mutations that can be used alone or in combination to enhance heteromultimer formation of the heteromultimers disclosed herein.

[0213] [Table 2]

[0214] In some embodiments, one or more residues that generate the CH3-CH3 interface in the fusion protein of the present application are replaced with charged amino acids, thereby making the interaction electrostatically unfavorable. For example, positively charged amino acids (e.g., lysine, arginine, or histidine) in the interface are replaced with negatively charged amino acids (e.g., aspartic acid or glutamic acid). Alternatively, or in combination with the above substitutions, negatively charged amino acids in the interface are replaced with positively charged amino acids. In certain embodiments, amino acids are replaced with non-naturally occurring amino acids with desired charge characteristics. It should be noted that mutation of negatively charged residues (Asp or Glu) to His increases the side chain volume, which may cause steric problems. Furthermore, His protein donor and acceptor types depend on the local environment. These issues should be considered with the design strategy. Because the interface residues are highly conserved in human and mouse IgG subclasses, the electrostatic induction effect disclosed herein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4. This strategy can also be extended to modifying uncharged residues in the CH3 domain interface to charged residues.

[0215] In part, the present disclosure provides for the desired pairing of asymmetric Fc-containing polypeptide chains using Fc sequences engineered to be complementary based on charge pairing (electrostatic attraction). One of the pair of electrostatically complementary Fc sequences can be optionally fused to a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide of a construct with or without an optional linker to generate a TGF-β superfamily type I, type II, or co-receptor receptor fusion polypeptide. This single chain can be co-expressed in a selected cell with an Fc sequence complementary to the first Fc to favor the generation of a desired multi-chain construct (e.g., a TGF-β superfamily heteromultimer). In this example based on electrostatic induction, SEQ ID NO: 140 (human G1Fc (E134K / D177K)) and SEQ ID NO: 141 (human G1Fc (K170D / K187D)) are examples of complementary Fc sequences (engineered amino acid substitutions are double underlined), and the TGF-β superfamily type I, type II, or co-receptor polypeptide of the construct can be fused to either SEQ ID NO: 140 or SEQ ID NO: 141 (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 understood that amino acid substitutions at the corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 4) will generate complementary Fc pairs that can be used in place of the complementary hG1Fc pair (SEQ ID NO: 140 and 141) below.

[0216] In part, the present disclosure provides for the desired pairing of asymmetric Fc-containing polypeptide chains using engineered Fc sequences for steric complementarity. In part, the present disclosure provides for knobs-into-holes pairing as an example of steric complementarity. One of the pair of sterically complementary Fc sequences can be optionally fused with a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide of a construct with or without an optional linker to generate a TGF-β superfamily type I, type II, or co-receptor fusion polypeptide. This single chain can be co-expressed in a selected cell with an Fc sequence complementary to the first Fc to advantageously generate a desired multi-chain construct. In this example based on ridge-indentation pairing, SEQ ID NO: 142 (human G1Fc(T144Y)) and SEQ ID NO: 143 (human G1Fc(Y185T)) are examples of complementary Fc sequences (engineered amino acid substitutions are double underlined), and the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide of the construct can be fused to either SEQ ID NO: 142 or SEQ ID NO: 143 (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 understood that amino acid substitutions at the corresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 4) will generate complementary Fc pairs that can be used in place of the complementary hG1Fc pair (SEQ ID NO: 142 and 143) below.

[0217] Examples of Fc complementarity based on ridge-indentation pairing in combination with engineered disulfide bonds are disclosed in SEQ ID NO: 144 (hG1Fc(S132C / T144W)) and SEQ ID NO: 145 (hG1Fc(Y127C / T144S / L146A / Y185V)). The engineered amino acid substitutions in these sequences are double underlined, and constructs can be fused to either SEQ ID NO: 144 or SEQ ID NO: 145 (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 Figure 4) will generate complementary Fc pairs that can be used in place of the complementary hG1Fc pair (SEQ ID NOs: 144 and 145) below.

[0218] In part, the present disclosure relates to human IgG and IgA C H Methods provide for the desired pairing of asymmetric Fc-containing polypeptide chains using engineered Fc sequences to create three-domain interdigitating β-strand segments. Such methods include strand-exchange engineered domain (SEED) sequences, which allow for the formation of SEEDbody fusion proteins. H Examples of suitable heterodimers include the use of 3 heterodimers (see, for example, Davis et al (2010) Protein Eng Design Sel 23:195-202). One of the pair of Fc sequences with SEED body complementarity can be optionally fused to a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide of the construct with or without an optional linker to generate a TGF-β superfamily fusion polypeptide. This single chain can be co-expressed in a selected cell with an Fc sequence complementary to the first Fc to favor the generation of the desired multi-chain construct. In this example based on SEED body (Sb) pairing, SEQ ID NO: 146 (hG1Fc(Sb AG)) and SEQ ID NO: 147 (hG1Fc(Sb GA ) are examples of complementary IgG Fc sequences (amino acid substitutions engineered from IgA Fc are double underlined), and the TGF-β superfamily type I, type II, or co-receptor polypeptides of the construct can be fused to either SEQ ID NO: 146 or SEQ ID NO: 147 (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 the corresponding positions (see FIG. 4) in hG1Fc, hG2Fc, hG3Fc, or hG4Fc will generate Fc monomers that can be used in the complementary IgG-IgA pairs below (SEQ ID NOs: 146 and 147).

[0219] In part, this disclosure relates to Fc C HThe desired pairing of asymmetric Fc-containing polypeptide chains is provided using a cleavable leucine zipper domain linked to the C-terminus of the three domains. The linkage of the leucine zipper is sufficient to cause the preferential assembly of heterodimeric antibody heavy chains. See, for example, Wranik et al (2012) J Biol Chem 287:43331-43339. As disclosed herein, one of the pair of Fc sequences linked to a leucine zipper-forming chain can be optionally fused to a TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide of a construct with or without an optional linker to generate a TGF-β superfamily fusion polypeptide. This single chain can be co-expressed in a selected cell with an Fc sequence linked to a complementary leucine zipper-forming chain to favor the generation of a desired multi-chain construct. Proteolytic digestion of the construct with bacterial endoproteinase Lys-C after purification can release the leucine zipper domain, resulting in an Fc construct whose structure is identical to that of the native Fc. In this example based on leucine zipper pairing, SEQ ID NO: 148 (hG1Fc-Ap1 (acidic)) and SEQ ID NO: 149 (hG1Fc-Bp1 (basic)) are examples of complementary IgG Fc sequences (the engineered complementary leucine zipper sequences are underlined), and the TGF-β superfamily type I, type II, or co-receptor polypeptide or co-receptor polypeptide of the construct can be fused to either SEQ ID NO: 148 or SEQ ID NO: 149 (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 a leucine zipper-forming sequence (see FIG. 4) linked to hG1Fc, hG2Fc, hG3Fc, or hG4Fc, with or without an optional linker, will generate an Fc monomer that can be used in the leucine zipper-forming pair (SEQ ID NOs: 148 and 149) described below.

[0220] In a preferred embodiment, the TGF-β superfamily receptor polypeptide, including its heteromultimers and homomultimers, that is the subject of use according to the methods described herein is an isolated polypeptide complex. As used herein, an isolated protein (or protein complex) or polypeptide (or polypeptide complex) is one that is separated from components of its natural environment. In some embodiments, the heteromultimeric complex of the present disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). Methods for assessing antibody purity are well known in the art (e.g., Flatman et al., (2007) J. Chromatogr. B 1999, 1999, 1999). 848:79-87. In some embodiments, the heteromultimer preparations of the present disclosure are substantially free of TGF-β superfamily type I receptor polypeptide homomultimers, TGF-β superfamily type II receptor polypeptide homomultimers, and / or TGF-β superfamily co-receptor polypeptide homomultimers. For example, in some embodiments, the heteromultimer preparations comprise less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily type I receptor polypeptide homomultimers. In some embodiments, the heteromultimer preparations comprise less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily type II receptor polypeptide homomultimers. In some embodiments, the heteromultimer preparations comprise less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily co-receptor polypeptide homomultimers.In some embodiments, the heteromultimer preparation comprises less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily type I receptor polypeptide homomultimers and less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily co-receptor polypeptide homomultimers. In some embodiments, the heteromultimer preparation comprises less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily type II receptor polypeptide homomultimers and less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% TGF-β superfamily co-receptor polypeptide homomultimers.

[0221] In certain embodiments, the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, or co-receptor polypeptide, as well as their heteromultimeric complexes, of the present disclosure can be produced by a variety of techniques known in the art. For example, the polypeptides of the present disclosure can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (see, for example, Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, the polypeptides and complexes of the present disclosure (including their fragments or variants) can be produced recombinantly using a variety of expression systems (e.g., E. coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus), as is well known in the art. In further embodiments, modified or unmodified polypeptides of the disclosure can be generated by digestion of recombinantly produced full-length TGF-β superfamily type I receptor, type II receptor, and / or co-receptor polypeptides with, for example, trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites.

[0222] 3. Nucleic Acids Encoding TGF-β Superfamily Type I Receptor Polypeptides, Type II Receptor Polypeptides, and Co-receptor Polypeptides In certain embodiments, the present disclosure provides isolated and / or recombinant nucleic acids encoding the TGF-β superfamily type I receptor, type II receptor, and co-receptor disclosed herein, including their fragments, functional variants, and fusion proteins. For example, SEQ ID NO: 13 encodes the naturally occurring human ActRIIA precursor polypeptide, while SEQ ID NO: 14 encodes the processed extracellular domain of ActRIIA. The nucleic acids of interest can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in the method for making the TGF-β superfamily heteromultimers of the present disclosure.

[0223] As used herein, an isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0224] In certain embodiments, the nucleic acid encoding the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide of the present disclosure is selected from the group consisting of SEQ ID NOs: 7, 8, 13, 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 , 85, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210, and variants thereof. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions, including allelic variants, and thus include SEQ ID NOs: 7, 8, 13, 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 2 , 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210.

[0225] In certain embodiments, the TGF-β superfamily type I receptor polypeptide, type II receptor polypeptide, and / or co-receptor polypeptide of the present disclosure is selected from the group consisting of SEQ ID NOs: 7, 8, 13, 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 109, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 8, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210. Those skilled in the art will recognize the sequences of SEQ ID NOs: 7, 8, 13, 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 129, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 222, 224, 226, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 2 It will be understood that 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 the sequence complementary to 28, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210 also fall within the scope of the present disclosure. In further embodiments, the nucleic acid sequences of the present disclosure may be isolated, recombinant, and / or fused to a heterologous nucleotide sequence, or may be nucleic acid sequences in a DNA library.

[0226] In other embodiments, the nucleic acids of the disclosure include those sequences set forth in SEQ ID NOs: 7, 8, 13, 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210; Also included are nucleotide sequences that hybridize under highly stringent conditions to the complements of 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210, or fragments thereof. Those skilled in the art will readily appreciate that suitable stringency conditions that promote DNA hybridization can vary. For example, hybridization at 6.0x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at 2.0x SSC at 50°C, can be performed. For example, the salt concentration during the washing step can be selected between low stringency at about 2.0x SSC at 50°C to high stringency at about 0.2x SSC at 50°C. In addition, the temperature of the washing 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 can be varied, or the other variable can be altered while the temperature or salt concentration is kept constant. In one embodiment, the present disclosure provides nucleic acids hybridized under low stringency conditions at 6x SSC at room temperature, followed by washing at 2x SSC at room temperature.

[0227] Also within the scope of this disclosure are isolated nucleic acids that differ from the nucleic acids as set forth in SEQ ID NOs: 7, 8, 13, 14, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 179, 183, 185, 186, 191, 194, 195, 200, 203, and 210 due to the degeneracy of the genetic code. For example, many amino acids are designated by two or more triplet bases. Codons that specify the same amino acid, or synonyms (e.g., CAU and CAC are synonyms for histidine), may result in "silent" mutations that do not affect the amino acid sequence of a pro...

Claims

1. A fusion polypeptide for use in the treatment or prevention of renal fibrosis, renal inflammation, and / or renal damage in a subject, the fusion polypeptide comprising (i) an ActRIIA polypeptide and (ii) an activin receptor-like kinase 4 (ALK4) polypeptide.

2. The fusion polypeptide for use according to claim 1, wherein the ActRIIA polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

12.

3. The fusion polypeptide for use according to claim 1, wherein the ActRIIA polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

11.

4. A fusion polypeptide for use as described in claim 1 or 2, wherein the ActRIIA polypeptide comprises the amino acid sequence of SEQ ID NO:

12.

5. A fusion polypeptide for use as described in claim 1 or 3, wherein the ActRIIA polypeptide comprises the amino acid sequence of SEQ ID NO:

11.

6. The fusion polypeptide for use according to any one of claims 1 to 5, wherein the fusion polypeptide further comprises an immunoglobulin Fc domain.

7. The fusion polypeptide for use according to claim 6, wherein the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 135 to 149.

8. 2. The fusion polypeptide for use according to claim 1, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 177 and 180.

9. 9. The fusion polypeptide for use according to claim 1 or 8, wherein the fusion polypeptide consists of an amino acid sequence selected from SEQ ID NO: 177, SEQ ID NO: 180, SEQ ID NO: 177 without a terminal lysine, and SEQ ID NO: 180 without a terminal lysine.

10. The fusion polypeptide for use according to any one of claims 1 to 9, wherein the ALK4 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

58.

11. The fusion polypeptide for use according to any one of claims 1 to 10, wherein the ALK4 polypeptide comprises the amino acid sequence of SEQ ID NO:

58.

12. A fusion polypeptide for use according to any one of claims 1 to 11, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 204 and 208.

13. 13. The fusion polypeptide for use according to any one of claims 1 to 12, wherein said fusion polypeptide consists of an amino acid sequence selected from SEQ ID NO: 204, SEQ ID NO: 208, SEQ ID NO: 204 without a terminal lysine, and SEQ ID NO: 208 without a terminal lysine.

14. A fusion polypeptide for use according to any one of claims 1 to 13, further comprising one or more linker sequences.

15. 15. The fusion polypeptide for use according to claim 14, wherein the one or more linkers are selected from TGGG (SEQ ID NO: 217), SGGG (SEQ ID NO: 218), TGGGG (SEQ ID NO: 219), SGGGG (SEQ ID NO: 220), GGGGS (SEQ ID NO: 221), GGGG (SEQ ID NO: 222), and GGG (SEQ ID NO: 223).

16. A fusion polypeptide for use according to any one of claims 1 to 15, wherein the ActRIIA polypeptide comprises the extracellular domain of ActRIIA and the ALK4 polypeptide comprises the extracellular domain of ALK4.

17. The fusion polypeptide for use according to any one of claims 1 to 16, wherein the fusion polypeptide is an ActRIIA-Fc-ALK4-Fc heterodimer comprising an ActRIIA-Fc polypeptide and an ALK4-Fc polypeptide.

18. 18. The fusion polypeptide for use according to any one of claims 1 to 7 and 17, wherein the Fc domain comprises one or more amino acid modifications that promote specific binding of the Fc domain to another Fc domain having the same or at least one different amino acid modification.

19. The fusion polypeptide for use according to claim 17 or 18, wherein the ActRIIA-Fc-ALK4-Fc heterodimer has increased binding activity to one or more of activin A, GDF11, and BMP6 compared to the ActRIIA-Fc homodimer, and / or has increased binding activity to one or more of GDF8, GDF11, and activin A compared to the ALK4-Fc homodimer.

20. The fusion polypeptide for use according to any one of claims 17 to 19, wherein the ActRIIA-Fc-ALK4-Fc heterodimer has increased binding activity to activin A and activin AC, and decreased binding activity to activin B, BMP7 and BMP10, compared to the ActRIIA-Fc homodimer.

21. The fusion polypeptide for use according to any one of claims 1 to 20, wherein said use results in a reduction of renal fibrosis, renal inflammation and / or renal damage in a subject in need thereof.

22. 22. The fusion polypeptide for use according to claim 21, wherein the renal fibrosis, renal inflammation and / or renal damage is Alport syndrome, focal segmental glomerulosclerosis or focal segmental glomerulonephritis.

23. A fusion polypeptide for use in treating or preventing cardiovascular disease in a subject, the fusion polypeptide comprising (i) an ActRIIA polypeptide, and (ii) an activin receptor-like kinase 4 (ALK4) polypeptide.

24. Therapeutic use of a fusion polypeptide according to any one of claims 1 to 20.

Images