Activin receptor type IIB variants and uses thereof

Abstract

There remains a need for effective therapeutic agents for the treatment of TGFβ superfamily-associated disorders. [Solution] Polypeptides comprising an activin receptor type IIB (ActRIIB) ectodomain (ECD) variant are provided. In some embodiments, the polypeptides of the present disclosure comprise an ActRIIB-ECD variant fused to an Fc domain portion. The present disclosure also provides pharmaceutical compositions and methods using the polypeptides to treat diseases and conditions associated with TGF-β superfamily ligand signaling, such as metabolic disorders, diabetes, obesity, cardiometabolic disease, pulmonary hypertension, fibrosis, muscle weakness and atrophy, bone damage, and / or low red blood cell levels (e.g., anemia).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international PCT application claiming priority to U.S. Provisional Patent Application No. 63 / 434,576, filed December 22, 2022, U.S. Provisional Patent Application No. 63 / 443,015, filed February 2, 2023, U.S. Provisional Patent Application No. 63 / 500,881, filed May 8, 2023, U.S. Provisional Patent Application No. 63 / 507,638, filed June 12, 2023, and U.S. Provisional Patent Application No. 63 / 515,562, filed July 25, 2023, the contents of each of which are incorporated herein by reference.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (35PH_003_05WO_SeqList_ST26.xml; size: 440,400 bytes; and creation date: December 18, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to polypeptides comprising activin receptor type IIB (ActRIIB) ectodomain (ECD) variants and their use for binding and neutralizing TGFβ superfamily ligands, particularly for the treatment of diseases and conditions associated with TGFβ superfamily signaling, such as metabolic disorders, cardiometabolic diseases, pulmonary hypertension, fibrosis, muscle weakness and atrophy, bone damage, and low red blood cell levels. [Background technology]

[0004] The transforming growth factor beta (TGFβ) superfamily contains 35 ligands that regulate several physiological processes, including cell proliferation, migration and differentiation, muscle growth, vascular homeostasis, and bone development. Disturbances in their levels and / or signaling pathways can result in significant pathological effects. For example, TGFβ and activin ligands are involved in the pathogenesis of multiple human diseases and play important pathogenic roles in many diseases. Examples of TGFβ superfamily-associated disorders include metabolic and cardiometabolic disorders (diabetes and obesity), pulmonary hypertension (including pulmonary arterial hypertension), hematological malignancies, solid tumors, bone marrow failure states, muscle weakness, and a wide variety of disorders characterized by uncontrolled fibrosis, such as pulmonary, hepatic, renal, and cardiac fibrosis, and systemic sclerosis (SSc, also known as scleroderma) (Nanthakumar, D.B. et al., 2015; Meng, X.-M. et al., 2016). There remains a need in the art for effective therapeutic agents for the treatment of TGFβ superfamily-associated disorders. Summary of the Invention

[0005] Provided herein are activin receptor type IIB (ActRIIB)-ectodomain (ECD)-based traps with a tailored ligand specificity profile for binding and neutralizing TGFβ superfamily ligands, as well as pharmaceutical compositions and methods of use thereof in the treatment of diseases and conditions associated with or mediated by TGFβ superfamily signaling.

[0006] The ActRIIB-ECD trap provided herein comprises an ActRIIB-ECD variant fused to an Fc domain monomer that can function to assemble the two polypeptides together. The ActRIIB-ECD variants provided herein are designed to tailor ligand specificity to maximize therapeutic efficacy in a particular disease indication while minimizing adverse effects. The ActRIIB-ECD variants provided herein are constructed by introducing novel amino acid substitutions into the ActRIIB-ECD to prevent or reduce disruption of endogenous BMP-9 and / or BMP-10 signaling while maintaining and / or increasing neutralization of other TGFβ superfamily ligands, such as activin A, activin B, GDF-8, and / or GDF-11. Without wishing to be limited by theory, the goal of avoiding BMP-9 and / or BMP-10 signaling is based on the discovery that these ligands are important for maintaining vascular quiescence and homeostasis (Desroches-Castan, A. et al., 2022). Wild-type ActRIIB binds to BMP-9 and BMP-10, and therefore, ActRIIB-ECD-based traps have the potential to disrupt vascular homeostasis, which could lead to bleeding concerns. Supporting this concept, telangiectasia, nosebleeds, and gingival bleeding were observed in a clinical study of the non-mutated ActRIIB-ECD trap (called ACE-031) (Campbell, C. et al., 2017). It was suggested that these vascular effects may be due to inhibition of the BMP-9 pathway.

[0007] Preferred ActRIIB-ECD variants provided herein exhibit (1) similar or improved binding to activin A, activin B, GDF-8, and / or GDF-11 compared to wild-type ActRIIB (competing with endogenous activin receptors for ligand binding, allowing for reduced or inhibited receptor signaling upon stimulation by endogenous ligands), and (2) reduced or eliminated binding to BMP-9 and / or BMP-10 compared to wild-type ActRIIB (allowing for the maintenance of constitutive BMP-9 and / or BMP-10 signaling). These variants can be used to treat diseases and conditions in which activin receptor signaling is elevated, such as pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or hybrid PH), metabolic diseases, bone diseases, muscle diseases, fibrosis, and / or low red blood cell levels (e.g., anemia). The variant may, for example, result in a reduction in the symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or hybrid PH), a reduction in bone resorption or osteoclast activity, an increase in bone formation or bone mineral density, an increase in muscle mass or strength, a reduction in fibrosis (e.g., a decrease in fibrosis or a slowing or halting of the progression of fibrosis), and / or an increase in red blood cell levels (e.g., an increase in hemoglobin levels, hematocrit, or red blood cell count).

[0008] In some embodiments, the present disclosure provides a polypeptide comprising an amino acid sequence at least 85% identical to an activin receptor type IIB (ActRIIB) ectodomain (ECD) variant, wherein the polypeptide comprises an amino acid substitution at a position corresponding to position 33 of SEQ ID NO: 2. In some embodiments, the ActRIIB ECD variant exhibits reduced inhibition of BMP-9 and / or BMP-10 compared to a wild-type ActRIIB ectodomain. In some embodiments, the amino acid substitution is selected from L33F, L33Q, L33Y, L33W, L33H, L33R, L33E, L33K, and L33M.

[0009] In some embodiments, the present disclosure provides a polypeptide comprising an amino acid sequence at least 90% identical to an activin receptor type IIB (ActRIIB) ectodomain (ECD) variant, the polypeptide comprising an amino acid substitution at a position corresponding to position 33 of SEQ ID NO: 2. In some embodiments, the ActRIIB ECD variant exhibits reduced inhibition of BMP-9 and / or BMP-10 compared to a wild-type ActRIIB ectodomain. In some embodiments, the amino acid substitution is selected from L33F, L33Q, L33Y, L33W, L33H, L33R, L33E, L33K, and L33M.

[0010] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33F. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0011] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33Q. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:11, or (b) comprises or consists of the amino acid sequence of SEQ ID NO:11.

[0012] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33Y. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 12, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0013] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33W. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 13, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0014] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33H. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 14, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0015] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33R. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 15, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0016] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33E. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 16, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0017] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33K. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 17, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0018] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33M. In some embodiments, the ActRIIB ECD variant (a) is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18, or (b) comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0019] In some embodiments, the AtRIIB ECD variant further comprises an amino acid substitution at position 27 of SEQ ID NO: 2. In some embodiments, the AtRIIB ECD variant further comprises an amino acid substitution at position 69 of SEQ ID NO: 2. In some embodiments, the AtRIIB-ECD variant further comprises one or more amino acid substitutions that are G27D, G27E, T69E, T69Q, or T69H.

[0020] In some embodiments, the ActRIIB-ECD variant further comprises one or more additional amino acids at the N-terminus or C-terminus: C. In some embodiments, the ActRIIB ECD variant further comprises the following amino acid at the N-terminus: GRGEA (SEQ ID NO: 63) and / or the following amino acid at the C-terminus: APT.

[0021] In some embodiments, the polypeptide further comprises an Fc domain monomer. In some embodiments, the polypeptide further comprises a peptide linker positioned between the ActRIIB ECD variant and the Fc domain monomer. In some embodiments, the polypeptide comprises the following structure from N-terminus to C-terminus: ActRIIB ECD-peptide linker-Fc domain monomer.

[0022] In some embodiments, the Fc domain monomer is an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc domain monomer is a human Fc domain monomer or a mouse Fc domain monomer.

[0023] In some embodiments, the Fc domain monomer is engineered to reduce aggregation or modulate the stability of a dimeric polypeptide. In some embodiments, the Fc domain monomer comprises amino acid substitutions of M252Y, S254T, and T256E (YTE). In some embodiments, the Fc domain monomer comprises an amino acid substitution of M252Y. In some embodiments, the Fc domain monomer comprises a D at position 356 and an L (DL) at position 358. In some embodiments, the Fc domain monomer comprises an E at position 356 and an M (EM) at position 358. In some embodiments, the Fc domain monomer further comprises a lysine residue (K) at the C-terminus.

[0024] In some embodiments, the Fc domain monomer comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 252-292. In some embodiments, the Fc domain monomer comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 252-292. In some embodiments, the Fc domain monomer is of the IgG1 isotype. In some embodiments, the Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 253, SEQ ID NO: 255, or SEQ ID NO: 256.

[0025] In some embodiments, the Fc domain monomers form dimers.

[0026] In some embodiments, the peptide linker is glycine-rich. In some embodiments, the peptide linker is 10 to 40 amino acids in length. In some embodiments, the linker is at least 10 amino acids in length, at least 14 amino acids in length, at least 19 amino acids in length, or at least 39 amino acids in length. In some embodiments, the linker is 10 amino acids in length, 14 amino acids in length, 19 amino acids in length, or 39 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 89, 94, or 98.

[0027] In some embodiments, ActRIIB-ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 4-62. In some embodiments, ActRIIB-ECD comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 4-62. In some embodiments, ActRIIB-ECD comprises or consists of an amino acid sequence of SEQ ID NOs: 10-18, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to this amino acid sequence. In some embodiments, ActRIIB-ECD comprises or consists of an amino acid sequence of SEQ ID NO: 13, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to this amino acid sequence. In some embodiments, the polypeptide comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 174-251. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 174-251.

[0028] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234.

[0029] In some embodiments, a polypeptide comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 211 and 230-234. In some embodiments, a polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 211 and 230-234. In some embodiments, a polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 231, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to this amino acid sequence. In some embodiments, a polypeptide of the present disclosure comprises or consists of the amino acid sequence of SEQ ID NO: 231. In some embodiments, a polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 234, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to this amino acid sequence. In some embodiments, a polypeptide of the present disclosure comprises or consists of the amino acid sequence of SEQ ID NO: 234.

[0030] In some embodiments, the polypeptide further comprises an albumin-binding domain, a fibronectin domain, or a human serum albumin domain fused to the N-terminus or C-terminus of ActRIIB-ECD via a linker. In some embodiments, the polypeptide further comprises a signal peptide of SEQ ID NO: 1 at the N-terminus of ActRIIB-ECD. In some embodiments, the signal peptide is cleaved from the mature protein.

[0031] In some embodiments, the polypeptide is conjugated to a targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety. In some embodiments, the targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety comprises an antibody or antigen-binding fragment thereof, a binding agent with affinity for another member of the TGFβ superfamily or for another therapeutic target, a radiotherapeutic agent, an imaging agent, a fluorescent moiety, a cytotoxic agent, an antimitotic agent, a nanoparticle-based carrier, a polymeric binding agent, a nanocarrier, an imaging agent, a stabilizing agent, a drug, a nanocarrier, or a dendrimer.

[0032] In some embodiments, the polypeptides form a dimer comprising a first polypeptide and a second polypeptide linked by at least one disulfide bond between an Fc domain monomer of the first polypeptide and an Fc domain monomer of the second polypeptide.

[0033] In some embodiments, the present disclosure provides a TGFβ superfamily ligand binding agent comprising a first polypeptide described herein and a second polypeptide described herein, wherein the first polypeptide and the second polypeptide are linked by at least one disulfide bond between an Fc domain monomer of the first polypeptide and an Fc domain monomer of the second polypeptide.

[0034] In some embodiments, the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 174-251, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to this amino acid sequence.

[0035] In some embodiments, the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to this amino acid sequence.

[0036] In some embodiments, the first and second polypeptides comprise or consist of an amino acid sequence selected from SEQ ID NOs:211 and 230-234, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to this amino acid sequence. In some embodiments, the first and second polypeptides comprise or consist of SEQ ID NO:231, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:231. In some embodiments, the first and second polypeptides comprise or consist of SEQ ID NO:234, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:234. In some embodiments, the first and second polypeptides comprise or consist of SEQ ID NO:211, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:211.

[0037] In some embodiments, the polypeptide and / or binding agent exhibits similar or increased binding to human activin A, activin B, GDF-8, and / or GDF-11 and decreased binding to human BMP-9 and / or BMP-10 compared to a polypeptide comprising wild-type ActRIIB-ECD. In some embodiments, the polypeptide and / or binding agent does not substantially bind to human BMP-9.

[0038] In some embodiments, the polypeptide and / or binding agent exhibits decreased binding to human BMP-10 compared to a polypeptide comprising wild-type ActRIIB-ECD. In some embodiments, the polypeptide and / or binding agent inhibits signaling of one or more of human activin A, activin B, GDF-8, and GDF-11. In some embodiments, the polypeptide or binding agent does not substantially inhibit signaling of human BMP-9 and / or BMP-10.

[0039] In some embodiments, the polypeptides and / or binding agents have an inhibitory potency against human BMP-9 and / or BMP-10 that is reduced by about 5-fold, about 10-fold, or about 100-fold or more compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 and / or BMP-10 signaling.

[0040] In some embodiments, the polypeptides and / or binding agents have an inhibitory potency against human BMP-9 and / or BMP-10 that is reduced by about 200-fold, about 300-fold, or more compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 and / or BMP-10 signaling.

[0041] In some embodiments, the polypeptides and / or binding agents have an inhibitory potency against human BMP-9 and / or BMP-10 that is about 5-fold, about 10-fold, or about 100-fold or more reduced compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against human BMP-9 and / or BMP-10 signaling.

[0042] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency against human BMP-9 and / or BMP-10 that is about 200-fold, or about 300-fold, or more reduced compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO:171.

[0043] In some embodiments, the polypeptides and / or binding agents have inhibitory potency against the same respective ligand(s) that is the same as or substantially the same as the inhibitory potency of human wild-type ActRIIB-ECD against one or more of human activin A, activin B, GDF-8, and GDF-11.

[0044] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency against the same respective ligand(s) that is the same as or substantially the same as the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 57 against one or more of human activin A, activin B, GDF-8, and GDF-11.

[0045] In some embodiments, the polypeptides and / or binding agents have increased inhibitory potency against one or more of human activin A, activin B, GDF-8, and GDF-11 relative to their respective ligand(s), and / or have reduced relative inhibitory potency against BMP-9 and / or BMP-10 relative to the inhibitory potency of human wild-type ActRIIB-ECD against the same ligands.

[0046] In some embodiments, the polypeptides and / or binding agents have increased inhibitory potency against one or more of human activin A, activin B, GDF-8, and GDF-11 relative to their respective ligand(s) compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the respective ligand(s), and / or have reduced relative inhibitory potency against BMP-9 and / or BMP-10 compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same ligand.

[0047] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency against one or more of human activin A, activin B, GDF-8, and GDF-11 that is increased by about 2-fold or more, about 3-fold or more, about 4-fold or more, or about 5-fold or more compared to the inhibitory potency of human wild-type ActRIIB-ECD or a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same respective ligand(s).

[0048] In some embodiments, the polypeptide and / or binding agent has a relative inhibitory potency against activin A that is at least about 2-fold higher than a polypeptide having the amino acid sequence set forth in SEQ ID NO: 57, and a relative inhibitory potency against BMP-9 and / or BMP-10 that is at least about 10-fold lower than a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.

[0049] In some embodiments, the polypeptide and / or binding agent (a) has an inhibitory potency against activin A that is at least about 5-fold greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the inhibitory potency of the polypeptide against BMP-9 and / or BMP-10 is at least about 100-fold less than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171; (b) has an inhibitory potency against activin B that is at least about 5-fold greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the inhibitory potency of the polypeptide against BMP-9 and / or BMP-10 is at least about 100-fold less than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171; or (c) has an inhibitory potency against both activin A and activin B that is at least about 5-fold greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the inhibitory potency of the polypeptide against BMP-9 and / or BMP-10 is at least about 100-fold less than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.

[0050] In some embodiments, the disclosure provides a nucleic acid molecule encoding a polypeptide described herein. In some embodiments, the nucleic acid further comprises the sequence set forth in SEQ ID NO: 297 at the 5' end of the nucleic acid molecule.

[0051] In some embodiments, the present disclosure provides vectors comprising the nucleic acid molecules described herein.

[0052] In some embodiments, the present disclosure provides a host cell comprising a nucleic acid molecule or vector described herein, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0053] In some embodiments, the present disclosure provides methods for preparing a polypeptide described herein, comprising: (a) providing a host cell comprising a nucleic acid molecule or vector described herein; (b) culturing the host cell under conditions that allow expression of the polypeptide; and (c) recovering the expressed polypeptide from the culture.

[0054] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a polypeptide or binding agent described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition is formulated for administration by injection or infusion. In some embodiments, the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

[0055] In some embodiments, the polypeptide or binding agent does not cause a vascular complication in the subject and / or does not increase vascular permeability or leakage in the subject, hi some embodiments, the polypeptide or binding agent does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and / or does not cause a hematologic complication in the subject.

[0056] In some embodiments, the present disclosure provides kits comprising a polypeptide, binding agent, or pharmaceutical composition described herein, and optionally, instructions for use.

[0057] In some embodiments, the present disclosure provides a method of treating or preventing a disease or condition associated with TGFβ superfamily ligand signaling in a subject in need thereof, the method comprising administering to the subject a polypeptide, binding agent, or pharmaceutical composition described herein. In some embodiments, the subject is a human. In some embodiments, the TGFβ superfamily ligand is one or more of activin A, activin B, GDF-8, and GDF-11.

[0058] In some embodiments, the present disclosure provides methods of treating or preventing a disease or condition mediated by activin A, activin B, GDF-8, and / or GDF-11 in a subject, the method comprising administering to the subject a polypeptide, binding agent, or pharmaceutical composition described herein. In some embodiments, the disease or condition is characterized by overexpression or overactivation of activin A and / or activin B and / or GDF-8 and / or GDF-11. In some embodiments, the disease or condition is selected from pulmonary hypertension (PH), fibrosis, muscle weakness or atrophy, metabolic disorders, cardiometabolic disease, bone damage, and low red blood cell levels.

[0059] In some embodiments, the PH is pulmonary arterial hypertension (PAH). In some embodiments, the PAH is idiopathic PAH, hereditary PAH, or PAH associated with infection, congenital heart abnormality, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, chronic obstructive pulmonary disease, autoimmune disorders (e.g., scleroderma or lupus), or drug use (e.g., cocaine or methamphetamine use). In some embodiments, the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, kidney fibrosis, myelofibrosis, systemic sclerosis, dermal fibrosis, cardiac fibrosis, myelofibrosis, corneal fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthrofibrosis, arthrofibrosis, tissue fibrosis, fibromatous proliferative disorder, or connective tissue disorder. In some embodiments, the muscle wasting or atrophy disease or condition is Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. In some embodiments, the metabolic disorder is obesity, type 1 diabetes, type 2 diabetes, or prediabetes. In some embodiments, the metabolic disorder is obesity. In some embodiments, the cardiometabolic disease or condition is heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF). In some embodiments, the bone damage comprises bone demineralization, osteoporosis (e.g., primary or secondary), osteopenia, osteopetrosis, fracture, bone loss associated with bone cancer or cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with treatment for obesity, low gravity-related bone loss, or immobility-related bone loss. In some embodiments, the disease or condition of low blood cell levels is anemia or blood loss.

[0060] In some embodiments, the present disclosure provides a method of reducing or inhibiting activin A, activin B, GDF-8, and / or GDF-11 signaling in a subject in need thereof, without substantially reducing or inhibiting BMP-9 and / or BMP-10 signaling in the subject, the method comprising administering to the subject a polypeptide, binding agent, or pharmaceutical composition described herein. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the method does not cause vascular complications in the subject, does not increase vascular permeability or leakage in the subject, does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and / or does not cause hematologic complications in the subject.

[0061] Further scope, applicability, and advantages of the present technology will become apparent from the non-limiting detailed description given below. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the present technology, is provided by way of example only, with reference to the accompanying drawings.

[0062] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0063] For a better understanding of the technology, and to show more clearly how it may be put into practice, reference is now made, by way of example, to the accompanying drawings which illustrate aspects and features according to non-limiting embodiments of the technology. [Brief explanation of the drawings]

[0064] [Figure 1A]

[0049] Figure 1 shows polyacrylamide gel electrophoresis analysis of representative ActRIIB-ECD polypeptide constructs under non-reducing conditions. After expression and purification, 1 μg of each protein was loaded onto the gel as indicated: P75: protein 75, P739: protein 739, P750: protein 750, P751: protein 751, P753: protein 753, P754: protein 754, P1182: protein 1182, P1185: protein 1185, P1229: protein 1229, P1371: protein 1371, P1372: protein 1372, P1373: protein 1373, P1374: protein 1374, P1375: protein 1375, P1389: protein 1389, P1406: protein 1406, and P1409: protein 1409. "NR": non-reducing conditions, "R": reducing conditions. [Figure 1B]

[0039] Figure 1 shows polyacrylamide gel electrophoresis analysis under reducing conditions of representative ActRIIB-ECD polypeptide constructs. After expression and purification, 1 μg of each protein was loaded onto the gel as indicated: P75: protein 75, P739: protein 739, P750: protein 750, P751: protein 751, P753: protein 753, P754: protein 754, P1182: protein 1182, P1185: protein 1185, P1229: protein 1229, P1371: protein 1371, P1372: protein 1372, P1373: protein 1373, P1374: protein 1374, P1375: protein 1375, P1389: protein 1389, P1406: protein 1406, and P1409: protein 1409. "NR": non-reducing conditions, "R": reducing conditions. [Figure 2A]As shown in the figure, a comparative chart is shown showing the IC50 values ​​of neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) for exemplary test proteins. Points in the center of the chart indicate low neutralization potency (high IC50 value) for a particular cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 value) for a particular cytokine. Exemplary test proteins shown in the figure are P739, P750, and P751. All drugs are compared to wild-type ActRIIB-ECD (P75). [Figure 2B] As shown in the figure, a comparative chart is shown showing the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) for exemplary test proteins. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a particular cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a particular cytokine. Exemplary test proteins shown in the figure are P753, P754, and P1229. All agents are compared to wild-type ActRIIB-ECD (P75). [Figure 2C] As shown in the figure, a comparative chart is shown showing the IC50 values ​​of neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) for exemplary test proteins. Points in the center of the chart indicate low neutralization potency (high IC50 value) for a particular cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 value) for a particular cytokine. Exemplary test proteins shown in the figure are P1373, P1371, and P1375. All drugs are compared to wild-type ActRIIB-ECD (P75). [Figure 2D]As shown in the figure, a comparative chart is shown showing the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) for exemplary test proteins. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a particular cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a particular cytokine. Exemplary test proteins shown in the figure are P1182 and P1185. All agents are compared to wild-type ActRIIB-ECD (P75). [Figure 2E] As shown in the figure, a comparative chart is shown showing the IC50 values ​​of neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) for exemplary test proteins. Points in the center of the chart indicate low neutralization potency (high IC50 value) for a particular cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 value) for a particular cytokine. Exemplary test proteins shown in the figure are P1389, P1406, and P1409. All drugs are compared to wild-type ActRIIB-ECD (P75). [Figure 2F] Representative results of a HEK-Blue cell-based assay for inhibition of activin A are shown for exemplary proteins P75, P1229, P1371, P1372, P1373, P1374, and P1375. [Figure 2G] Representative results of a HEK-Blue cell-based assay for inhibition of activin B are shown for exemplary proteins P75, P1229, P1371, P1372, P1373, P1374, and P1375. [Figure 2H] Representative results of a HEK-Blue cell-based assay for inhibition of GDF-8 are shown for exemplary proteins P75, P1229, P1371, P1372, P1373, P1374, and P1375. [Figure 2I]Representative results of a HEK-Blue cell-based assay for inhibition of GDF-11 are shown for exemplary proteins P75, P1229, P1371, P1372, P1373, P1374, and P1375. [Figure 2J] Representative results of a HEK-Blue cell-based assay for the inhibition of BMP-9 are shown for exemplary proteins P75, P1229, P1372, P1373, P1374, and P1375. [Figure 2K] Representative results of a HEK-Blue cell-based assay for the inhibition of BMP-10 are shown for exemplary proteins P75, P1229, P1372, P1373, P1374, and P1375. [Figure 2L] Representative results of a HEK-Blue cell-based assay for inhibition of activin A are shown for exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486. [Figure 2M] Representative results of a HEK-Blue cell-based assay for inhibition of activin B are shown for exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486. [Figure 2N] Representative results of a HEK-Blue cell-based assay for inhibition of GDF-8 are shown for exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486. [Figure 2O] Representative results of a HEK-Blue cell-based assay for inhibition of GDF-11 are shown for exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486. [Figure 2P] Representative results of a HEK-Blue cell-based assay for the inhibition of BMP-9 are shown for exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486. ​​Error bars indicate the standard error of the mean (SEM). [Figure 2Q]Representative results of a HEK-Blue cell-based assay for the inhibition of BMP-10 are shown for exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486. ​​Error bars indicate the standard error of the mean (SEM). [Figure 3]

[0039] Figure 1 shows the results of an ELISA for activin A using supernatant from small-scale production of ActRIIB-ECD fusion protein. Results are expressed as a percentage (%) of the signal obtained with wild-type ActRIIB-ECD-Fc(P75). In this assay, loss of signal indicates increased binding of the exemplary test agent to activin A. Error bars indicate the standard error of the mean (SEM). This graph was generated using GraphPad Prism 9.0. [Figure 4] Figure 1 shows the results of an ELISA for BMP-9 using supernatant from small-scale production of ActRIIB-ECD fusion protein. Results are expressed as a percentage (%) of the signal obtained with wild-type ActRIIB-ECD-Fc(P75). In this assay, loss of signal indicates reduced binding of the exemplary test agent to BMP-9. Error bars indicate the standard error of the mean (SEM). This graph was generated using GraphPad Prism 9.0. [Figure 5A] Figure 1 shows the results of an assay for BMP-10 using supernatant from small-scale production of ActRIIB-ECD fusion protein. The results of the cell-based assay are expressed as a percentage (%) of the signal obtained with BMP-10 alone. In this assay, loss of signal indicates increased binding of the exemplary test agent to BMP-10. Error bars indicate the standard error of the mean (SEM). These graphs were generated using GraphPad Prism 9.0. [Figure 5B] Figure 1 shows the results of an assay for BMP-10 using supernatant from small-scale production of ActRIIB-ECD fusion protein. Biolayer interferometry was used to derive KD values ​​for exemplary test agents. Error bars indicate the standard error of the mean (SEM). These graphs were generated using GraphPad Prism 9.0. [Figure 6] Results of a single-injection experiment in wild-type mice are shown. Male mice were injected with the indicated single dose of test agent (25 mg / kg, subcutaneously) and weight gain was assessed 4 days later. Results were normalized to vehicle control, and error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001 vs. vehicle group). [Figure 7A] Figure 1 shows weight gain in male mice injected (subcutaneously) with the test proteins. Results for P750 (1, 5, or 25 mg / kg) are shown. Injections were performed twice weekly for 11 days (P750) or 7 days (P1229 and P75). Results were normalized to vehicle control, and error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.001, ***p<0.0001 vs. vehicle group). [Figure 7B] Figure 1 shows weight gain in male mice injected (subcutaneously) with the test proteins. Results are shown for P1229 (5 or 25 mg / kg) and P75 (25 mg / kg). Injections were performed twice weekly for 11 days (P750) or 7 days (P1229 and P75). Results were normalized to vehicle control, and error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.001, ***p<0.0001 vs. vehicle group). [Figure 8] 1 shows an exemplary schematic diagram of a binder as described herein. [Figure 9A] Changes in skeletal muscle mass after one week of treatment with vehicle, P1229 (5 or 25 mg / kg), and P75 (25 mg / kg) are shown. Injections were administered subcutaneously twice weekly. The mean weight of the tibia anterior is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.001 vs. vehicle group). [Figure 9B] Changes in skeletal muscle mass after one week of treatment with vehicle, P1229 (5 or 25 mg / kg), and P75 (25 mg / kg) are shown. Injections were administered subcutaneously twice weekly. Tibia weights are shown normalized to the vehicle control. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.001 vs. vehicle group). [Figure 9C] Changes in skeletal muscle mass after one week of treatment with vehicle, P1229 (5 or 25 mg / kg), and P75 (25 mg / kg) are shown. Injections were administered subcutaneously twice weekly. The mean weight of the gastrocnemius muscle is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.001 vs. vehicle group). [Figure 9D] Changes in skeletal muscle mass after one week of treatment with vehicle, P1229 (5 or 25 mg / kg), and P75 (25 mg / kg) are shown. Injections were administered subcutaneously twice weekly. Gastrocnemius muscle weights are shown normalized to the vehicle control. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.001 vs. vehicle group). [Figure 10] Changes in Mss51 expression in the tibialis anterior muscle after one week of treatment with vehicle, P1229 (5 or 25 mg / kg), and P75 (25 mg / kg). Injections were administered subcutaneously twice weekly. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (****p<0.0001 vs. vehicle group). [Figure 11A]Changes in body composition in diet-induced obese (DIO) mice were subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, murine bimagrumab (an antibody targeting ActRIIB and AcRIIA), 20 mg / kg, once weekly). Mean lean body mass is shown. Each parameter was assessed by echoMRI. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05 vs. vehicle group). [Figure 11B] Changes in body composition in diet-induced obese (DIO) mice were subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, murine bimagrumab (an antibody targeting ActRIIB and AcRIIA), 20 mg / kg, once weekly). Mean fat mass is shown. Each parameter was assessed by echoMRI. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05 vs. vehicle group). [Figure 11C] Changes in body composition in diet-induced obese (DIO) mice were subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, murine bimagrumab (an antibody targeting ActRIIB and AcRIIA), 20 mg / kg, once weekly). The ratio of lean body mass to fat mass is shown. Each parameter was assessed by echoMRI. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05 vs. vehicle group). [Figure 11D]Changes in body composition in diet-induced obese (DIO) mice were subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, murine bimagrumab (an antibody targeting ActRIIB and AcRIIA), 20 mg / kg, once weekly). Mean lean body mass is shown. Each parameter was assessed by echoMRI. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05 vs. vehicle group). [Figure 11E] Changes in body composition in diet-induced obese (DIO) mice were subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, murine bimagrumab (an antibody targeting ActRIIB and AcRIIA), 20 mg / kg, once weekly). Mean fat mass is shown. Each parameter was assessed by echoMRI. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05 vs. vehicle group). [Figure 11F] Changes in body composition in diet-induced obese (DIO) mice were subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, murine bimagrumab (an antibody targeting ActRIIB and AcRIIA), 20 mg / kg, once weekly). The ratio of lean body mass to fat mass is shown. Each parameter was assessed by echoMRI. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05 vs. vehicle group). [Figure 12A] Food consumption over time is shown in DIO mice subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, or murine bimagrumab, 20 mg / kg, once weekly). [Figure 12B]Food consumption over time is shown in DIO mice subcutaneously injected with vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866, or murine bimagrumab, 20 mg / kg, once weekly). [Figure 12C] Skeletal muscle weights at the end of the study are shown. The weight of the tibialis anterior muscle is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01 vs. vehicle group). [Figure 12D] Skeletal muscle weights at the end of the study are shown. The weight of the tibialis anterior muscle is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01 vs. vehicle group). [Figure 12E] Skeletal muscle weights at the end of the study are shown. Gastrocnemius muscle weights are shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01 vs. vehicle group). [Figure 12F] Skeletal muscle weights at the end of the study are shown. Gastrocnemius muscle weights are shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01 vs. vehicle group). [Figure 12G] Skeletal muscle weights at the end of the study are shown. Soleus muscle weights are shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01 vs. vehicle group). [Figure 12H] Skeletal muscle weights at the end of the study are shown. Soleus muscle weights are shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, **p<0.01 vs. vehicle group). [Figure 13]Figure 1 shows changes in skeletal muscle gene expression in DIO mice 3 weeks after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Figures A and B show the expression of Mss51 in the gastrocnemius muscle. Figure C shows the change in the expression ratio of serpin1 to Id1 in the soleus muscle. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p < 0.05, ***p < 0.01, ***p < 0.001 vs. vehicle group). [Figure 14A] Figure 1 shows changes in hepatic gene expression in DIO mice after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Expression of the Ahsg gene, which encodes fetuin-A, is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p < 0.05, ***p < 0.01, ***p < 0.001 vs. vehicle group). [Figure 14B] Figure 1 shows changes in hepatic gene expression in DIO mice after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Expression of the Ahsg gene, which encodes fetuin-A, is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p < 0.05, ***p < 0.01, ***p < 0.001 vs. vehicle group). [Figure 14C]Figure 1 shows changes in hepatic gene expression in DIO mice after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Expression of the Fgf21 gene, which encodes FGFA, is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.01, ***p<0.001 vs. vehicle group). [Figure 14D] Figure 1 shows changes in hepatic gene expression in DIO mice after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Expression of the Fgf21 gene, which encodes FGFA, is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p<0.05, ***p<0.01, ***p<0.001 vs. vehicle group). [Figure 14E] Figure 1 shows changes in hepatic gene expression in DIO mice after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Expression of the Inhbe gene, which encodes activin E, is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p < 0.05, ***p < 0.01, ***p < 0.001 vs. vehicle group). [Figure 14F]Figure 1 shows changes in hepatic gene expression in DIO mice after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Expression of the Inhbe gene, which encodes activin E, is shown. Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (*p < 0.05, ***p < 0.01, ***p < 0.001 vs. vehicle group). [Figure 15] Panels A and B show the changes in follicle-stimulating hormone (FSH) expression in DIO mice 3 weeks after subcutaneous injection of vehicle, P1229 (25 mg / kg, twice weekly), P75 (25 mg / kg, twice weekly), P1373 (5, 20, or 50 mg / kg, twice weekly), or P1307 (CDD866), or murine bimagrumab (20 mg / kg, once weekly). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test (***p < 0.001, ****p < 0.0001 vs. vehicle group). DETAILED DESCRIPTION OF THE INVENTION

[0065] overview Activin type II receptors are single transmembrane domain receptors that regulate the signaling of ligands in the TGFβ superfamily. There are two types of activin type II receptors: ActRIIA and ActRIIB. Examples of TGFβ superfamily ligands include activins (e.g., activin A and activin B), inhibins, growth differentiation factors (GDFs) (e.g., GDF-8, also known as myostatin and GDF-11), and bone morphogenetic proteins (BMPs) (BMP-9, BMP-10). The activity of TGFβ superfamily ligands is involved in various diseases and disorders, including pulmonary hypertension (PH), fibrosis, muscle diseases (including muscular dystrophy), metabolic disorders (including type II diabetes), bone diseases, and anemia.

[0066] One approach to developing therapeutics that inhibit TGFβ superfamily ligand function has been to block access to cell surface receptors by using soluble decoy receptors (also called receptor ectodomain (ECD)-based ligand traps) that bind and sequester the ligand. In general, receptor ECD-based traps are a class of therapeutic agents that can selectively sequester ligands and can be optimized using protein engineering approaches. For example, polypeptide fusions based on the TGFβ receptor ectodomain that bind to or "trap" TGFβ1 and / or TGFβ2 and / or TGFβ3 ligand isoforms have been used to inhibit TGFβ signaling (e.g., WO01 / 83525, WO2005 / 028517, WO2008 / 113185, WO2008 / 157367, WO2010 / 0031168, WO2010 / 0031168). (See US2005 / 0203022, US2007 / 0244042, US8318135, US8658135, US8815247, US2015 / 0225483, US2015 / 0056199, and WO2017 / 037634).

[0067] In the pulmonary endothelium and vasculature, bone morphogenetic proteins (BMPs) can induce antiproliferative effects on smooth muscle cell (SMC) and endothelial cell (EC) survival, whereas activins and growth differentiation factors (GDFs) can induce opposing effects, namely, pro-proliferative effects on SMCs and apoptosis of ECs (Yung, LM et al., 2020; Ryanto, GR et al., 2021). Under physiological conditions, these ligands act in concert to maintain homeostasis. However, in certain disease states, such as PAH, these pathways become imbalanced. For example, approximately 80% of familial cases and approximately 20% of idiopathic cases of PAH are caused by mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) (Austin, ED and Loyd, JE, 2007; Quarck, R. and Perros, F., 2017). This leads to an imbalance between the activin / GDF and BMP signaling pathways (Ryanto, GRT et al., 2021). Therefore, it is desirable to provide a receptor ectodomain-based trap that can neutralize certain ligands but not others to rebalance the pathways and reestablish vascular homeostasis.

[0068] Thus, the present application provides TGFβ superfamily ligand binding agents that exhibit improved ligand binding profiles and therapeutic efficacy.

[0069] The present technology is described in more detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology can be implemented or all the features that can be added to the technology. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be omitted from that embodiment. Additionally, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, and such modifications and additions do not depart from the present technology. Thus, the following description is intended to illustrate some specific embodiments of the present technology, but is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0070] definition In order to provide a clear and consistent understanding of the terminology used herein, some definitions are provided below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0071] The use of the terms "a," "an," and "the," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Similarly, the term "another" can mean at least a second or more. These terms should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0072] As used herein, the terms "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "include" and "includes"), or "containing" (and any form of "containing", such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. The term "consisting of" is to be construed as close-ended.

[0073] The term "about" is used to indicate that a value or amount refers to the actual given value and approximations of such a given value that would be reasonably inferred based on common skill in the art, including equivalents and approximations based on experimental and / or measurement conditions for such a given value. For example, the term "about" in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0074] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of the specified features or components, regardless of the presence or absence of the others. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. The term "or," as used herein, is understood to be inclusive and encompasses both "or" and "and," unless specifically stated otherwise or clear from the context. For example, an embodiment of "a composition comprising A or B" would typically present an aspect having a composition containing both A and B. However, "or" should be interpreted to exclude those aspects that cannot be combined without contradiction (e.g., a composition pH of 9-10 or 7-8).

[0075] As used herein, terms such as "1 to 20" should be understood to include any individual value subsumed therein, inclusive of 1 and 20. Thus, the term "1 to 20" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20. Terms such as "1 to 20" also include any individual subranges subsumed therein, inclusive of 1 and 20. Thus, the term "1 to 20" also includes subranges such as "1 to 9," "2 to 9," "3 to 5," "5 to 9," "5 to 20," "8 to 20," etc. The same applies to similar expressions such as, but not limited to, "1 to 19," "1 to 18," "1 to 10," "1 to 9," "5 to 15," etc.

[0076] As used herein, terms such as "about 15 to about 35" should be understood to include any individual value between 15 and 35, inclusive. Thus, terms such as "about 15 to about 35" include any number between 15 and 35, inclusive, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and / or 35. Terms such as "about 15 to about 35" also include any individual subranges between 15 and 35, inclusive, such as "about 16 to about 34," "about 16 to about 24," "about 24 to about 34," etc. The term "about" in the context of a number of amino acids means that the particular number of amino acids is specifically included, allowing for a + / - 2 variation in the number of amino acid residues. Thus, terms such as "about 15 to about 35" also include "13 to 37," "13 to 35," "17 to 37," "17 to 35," etc. The same applies to similar expressions such as, but not limited to, "about 16 to about 34," "about 16 to about 24," and "about 24 to about 34."

[0077] As used herein, terms such as "at least 80% identical" are understood to include 80% and 100%, inclusive, and any individual value, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. The term "at least 80% identical" also includes any individual subranges therein, inclusive, such as "85% to 99%," "97% to 100%," and "90% to 100%." ​​The same applies to similar expressions, such as, but not limited to, "at least 70% identical," "at least 90% identical," and the like.

[0078] As used herein, the term "IC50" refers to the half-maximal inhibitory concentration (i.e., the concentration of a substance required for 50% inhibition in vitro). It is a measure of the potency or effectiveness of a substance in inhibiting a specific biological or biochemical function. IC50 values ​​are typically expressed as molar concentrations. The IC50 of an inhibitor can be determined by constructing a dose-response curve and examining the effect of different concentrations of the inhibitor on the specific biological or biochemical function in question.

[0079] As used herein, the term "inhibitory potency" refers to the effectiveness of a substance in inhibiting a specific biological or biochemical function, such as, but not limited to, the binding between a protein receptor and its ligand or the activation of a cellular receptor by its ligand. In some embodiments, inhibitory potency is determined by measuring the IC50 of the inhibitor for a specific ligand or substance. In this case, the relative inhibitory potency for different inhibitors and / or ligands can be assessed by comparing IC50 values. For example, a relative inhibitory potency of 3:1 means that the ratio of the IC50 values ​​of the two substances being compared is 3:1, where the first substance has a lower inhibitory potency (i.e., a higher IC50) than the second substance. A relative inhibitory potency of 1:3 means that the ratio of the IC50 values ​​of the two substances being compared is 1:3, where the first substance has a higher inhibitory potency (i.e., a lower IC50) than the second substance. Because the IC50 of an inhibitor can vary depending on the assay conditions, the relative inhibitory potency for different inhibitors and / or ligands is generally determined by comparing IC50 values ​​obtained under the same assay conditions. The terms "inhibition potency," "inhibitory potency," "inhibitory efficacy," and "neutralizing efficacy" are used interchangeably herein.

[0080] As used herein, the term "substantially the same" with respect to relative inhibitory potency means that two proteins have about the same relative inhibitory potency, e.g., no more than about 2-fold different (+ / - 2-fold), under the same experimental conditions, e.g., conditions where the ratio of the IC50 values ​​of the two proteins is about 2:1, 1:2, or 1:1.

[0081] As used herein, the term "functionally equivalent" refers to a variant sequence that has the same or substantially the same biological activity or function as the original sequence from which it is derived, e.g., a sequence that has no significant changes in physiological, chemical, physicochemical, or functional properties compared to the original sequence. The term "substantially identical" refers to a sequence that is functionally equivalent to and has a high degree of sequence identity with the original or reference sequence. Generally, a substantially identical sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the original or reference sequence and has the same function. In some cases, when referring to nucleic acid sequences, a substantially identical sequence hybridizes to the original sequence under high stringency conditions, e.g., salt and temperature conditions substantially equivalent to 0.5X SSC to about 5X SSC and 65°C for both hybridization and washing.

[0082] The term "dimer" refers to the presence of two polypeptides described herein in a TGFβ superfamily ligand binding agent (also referred to herein as a "binder"). A "homodimer" means that the two polypeptides have the same amino acid sequence, while a "heterodimer" means that the two polypeptides have different amino acid sequences.

[0083] The term "bivalent" refers to the presence of two TGFβR superfamily ligand binding regions (eg, ectodomains) in a TGFβ superfamily ligand binding agent.

[0084] As used herein, a "recombinant polypeptide" is a polypeptide made through the use of recombinant DNA technology or genetic engineering. In the context of this disclosure, a recombinant polypeptide is often referred to as a "polypeptide construct" or simply as a "polypeptide."

[0085] Proteins (including fragments thereof, preferably biologically active fragments, and peptides usually having fewer than 30 amino acids) comprise two or more amino acids coupled to each other via covalent peptide bonds (resulting in a chain of amino acids). As used herein, the term "polypeptide" describes a group of molecules usually consisting of more than 10 amino acids. The terms "polypeptide," "polypeptide chain," and "chain" are used interchangeably herein. Polypeptides can also form multimers, such as dimers, trimers, and higher oligomers, i.e., multimers consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers or trimers may or may not be identical. The corresponding higher-order structures of such multimers are consequently referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally modified peptides / polypeptides / proteins, e.g., modified by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. As referred to herein, a "peptide," "polypeptide," or "protein" may also be chemically modified, such as by pegylation. Such modifications are well known in the art and are described herein.

[0086] As used herein, the terms "(specifically) bind," "(specifically) recognize," "specific for," "(specifically) directed," and "(specifically) react" mean that a polypeptide interacts or specifically interacts with a given target(s), such as a specific member(s) of the TGFβ superfamily of ligands. Specific binding is believed to be influenced by specific motifs in the amino acid sequence of the polypeptide. Binding is thus achieved as a result of their primary, secondary, and / or tertiary structure and secondary modifications of that structure. Specific interaction between a target-interaction site and its specific target may result in simple binding of the site to the target. Furthermore, specific interaction between a target-interaction site and its specific target may alternatively or additionally result in the initiation of a signal or block the target from performing another activity, such as binding to an endogenous receptor, for example, due to induction of a conformational change in the target, oligomerization of the target, etc.

[0087] Generally, binding is considered specific when the binding affinity is about 10-12 to 10-9 M, 10-12 to 10-19 M, 10-11 to 10-9 M, or about 10-11 to 10-9 M. Whether a polypeptide or binding agent specifically reacts with or binds to a target can be readily tested, inter alia, by comparing the reaction of the polypeptide or binding agent with the target to the reaction of the polypeptide or binding agent with other proteins. In some embodiments, a polypeptide or binding agent of the present disclosure does not substantially bind to TGF-β superfamily ligands other than the desired ligand, e.g., does not substantially bind to BMP-9.

[0088] As used herein, the terms "does not substantially bind" or "cannot bind" mean that a polypeptide or binding agent of the disclosure exhibits no detectable binding to a given target, e.g., no more than 30%, no more than 20%, no more than 10%, or no more than 9%, 8%, 7%, 6%, 5%, or 3% reactivity with a given target.

[0089] As used herein, the term "selectively binds" refers to a polypeptide binding to a target site that is not shared with other proteins. Generally, a selective binding agent does not cross-react with other proteins and exclusively binds to a designated target protein(s). In the context of the present disclosure, "selective for activin A and GDF-8" means that a polypeptide or binding agent exclusively binds to or neutralizes activin A and GDF-8 ligands without substantially binding to or neutralizing other TGFβ superfamily ligands, such as BMP-9.

[0090] "Half-life" means the time it takes for 50% of an administered drug to be eliminated through biological processes, such as metabolism, excretion, etc.

[0091] "Hepatic first-pass metabolism" refers to the tendency of a drug to be metabolized upon first contact with the liver, i.e., during its first pass through the liver.

[0092] "Volume of distribution" refers to the degree of retention of a drug throughout the various compartments of the body, eg, intracellular and extracellular spaces, tissues and organs, and the distribution of the drug within these compartments.

[0093] "Extent of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins, such as albumin, resulting in a reduction or loss of the drug's biological activity.

[0094] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val), negatively charged side chains (e.g., Asp, Glu), positively charged side chains (e.g., Arg, His, Lys), or uncharged polar side chains (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0095] Similarly, "percent (%) nucleic acid sequence identity" with respect to a nucleic acid sequence of a polypeptide or binding agent identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the polypeptide or binding agent. A particular method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with the overlap span and overlap fraction set to 1 and 0.125, respectively.

[0096] TGFβ superfamily ligand binders In some embodiments, the present disclosure provides a TGFβ superfamily ligand binding agent comprising an ActRIIB-ECD region, a linker region, and an Fc domain (also referred to herein as a "binding agent" or "TGFβ ligand binding agent"). The individual components of the binding agents described herein are described in further detail in the following sections. In general, however, the binding agents described herein are dimeric proteins comprising two polypeptides, each comprising an ActRIIB-ECD, a peptide linker, and an Fc domain monomer. The two polypeptides assemble via the Fc domain monomer to form the dimeric binding agent described herein. See the schematic diagram in Figure 8. When assembled, the Fc domain monomers in each polypeptide form a dimeric Fc domain at one end and a bivalent ActRIIB-ECD region at the other end. The binding agents of the present disclosure can bind to one or more ligands selected from activin A, activin B, GDF-8, and GDF-11 and inhibit signaling through the receptors of the one or more ligands described above, without substantially binding to BMP-9 and / or BMP-10 and / or inhibiting BMP-9 and / or BMP-10 signaling through their receptors. The binding agents can also have additional biological activities or functions, such as binding to other ligands or targets, as further described herein.

[0097] In some embodiments, the binding agents of the present disclosure comprise two polypeptide chains associated via an antibody Fc domain monomer, or via a constant CH2 domain, a constant CH3 domain, and / or a combination of CH2 and CH3. The antibody constant region can be derived from, or substantially identical to, a human IgG1, IgG2, IgG3, or IgG4 antibody. Association of both polypeptide chains generally occurs during protein expression and secretion, e.g., in mammalian cells. The Fc domain monomer is generally of human origin and typically comprises a CH2, CH3, or CH2 and CH3 from an antibody heavy chain, which provides disulfide bridges between the single polypeptide chains. In one embodiment, the Fc domain monomer provides at least one disulfide bond between the single polypeptide chains. In another embodiment, the Fc domain monomer provides at least two disulfide bonds between the single polypeptide chains. In some cases, the antibody heavy chain also provides for Protein A-based isolation of the dimeric polypeptide, e.g., after production in a host cell.

[0098] As noted above, point mutations in certain TGFβ superfamily ligand binding agents and ECDs have been described in the art. See, e.g., WO2021 / 158675, WO2022 / 150590, WO2021 / 158675, WO2022 / 072882, WO2021 / 189019, and WO2021 / 189010. While point mutations in the ActRIIB ECD have been described in the context of other TGFβ superfamily ligand binding agents, the effect of these mutations in the context of these previously described agents is not predictive of the effect of these same mutations in the context of the binding agents described herein. See, e.g., PCT / CA2023 / 050116, which describes the unpredictability of point mutations in the ActRIIB ECD when combined with linkers of various lengths. Thus, the efficacy of certain binding agents described herein is determined not only by the mutations contained in the extracellular ligand-binding domain, but also by the length of the linker used. As shown herein, the length of the linker connecting the ActRIIB ECD to the Fc domain has unpredictable effects on binding to and inhibition of TGFβ superfamily ligands. Accordingly, the present application provides TGFβ superfamily ligand binders that exhibit improved ligand-binding profiles and therapeutic efficacy. These compounds are useful for treating a variety of diseases and disorders induced by TGFβ superfamily ligands, including pulmonary hypertension, muscle diseases, metabolic disorders, bone diseases, anemia, and fibrosis.

[0099] Additional ECD-based traps, such as luspatercept and sotatercept, are being clinically evaluated. Luspatercept (ACE-536, also known as REBLOZYL®) is a soluble fusion protein composed of a modified form of the extracellular domain of activin receptor type IIB (ActRIIB) linked to the Fc portion of human IgG1. Luspatercept inhibits several endogenous TGFβ superfamily ligands, thereby reducing Smad2 / 3 signaling. It is used to treat anemia in beta-thalassemia and myelodysplastic syndromes. For a description of luspatercept and other related fusion proteins, see, e.g., U.S. Patent Nos. 7,842,663, 8,058,229, 8,216,997, 8,252,900, 8,343,933, 8,361,957, 8,703,927, 9,138,459, 9,399,669, 9,439,945, 9,932,379, 10,131,700, 10,259,861, 10,689,427, and 10,829,532.

[0100] Sotatercept (also known as ACE-011) is a soluble decoy receptor composed of the extracellular domain of activin receptor type IIA (ActRIIA) linked to the Fc portion of human IgG1, capable of binding to and neutralizing activin and GDF. Sotatercept has been evaluated in healthy volunteers and patients with conditions characterized by dysfunctional TGF-β superfamily signaling, including hematologic disorders, bone loss, chemotherapy-induced anemia, multiple myeloma, myelodysplastic syndrome, β-thalassemia, and end-stage renal disease (Raftopoulos, H. et al., 2016; Abdulkadyrov, K. et al., 2014; Ruckle, J. et al., 2009; Komrokji, R. et al., 2018; Cappellini, MD et al., 2019; Coyne, D. et al., 2019; Sherman, M. et al., 2013). Recently, sotatercept has been evaluated for the treatment of pulmonary arterial hypertension (PAH).

[0101] By acting as a ligand trap for activins and GDFs, sotatercept may correct the imbalance between the growth-promoting activin / growth differentiation factor pathway and the growth-inhibitory BMP pathway that occurs in PAH. In a phase 2 study in PAH patients, sotatercept has been shown to reduce pulmonary vascular resistance (Humbert, M. et al., 2021). Additional PH studies, including phase 3 studies, are ongoing or planned. For a description of sotatercept and other related fusion proteins, see, e.g., U.S. Patent Nos. 7,612,041, 7,709,605, 7,951,771, 7,988,973, 8,007,809, 8,629,109, 8,895,016, and 9,163,075. However, in several clinical studies of sotatercept, vascular and hematologic side effects have been found to be dose-limiting and limit potential therapeutic efficacy. For example, a multiple ascending dose study in healthy postmenopausal women planned to evaluate four doses—0.1, 0.3, 1 mg / kg, and 2 mg / kg—but was terminated early at the 1 mg / kg level because increases in hemoglobin, hematocrit, and red blood cell count were found to be dose-limiting (Sherman, M. Let et al., 2013). In a phase 2 clinical trial in patients with PAH, thrombocytopenia and increased hemoglobin levels were the most common hematologic adverse events, with 17% of patients receiving the 0.7 mg / kg dose experiencing increased hemoglobin (Humbert, M. et al., 2021). Such vascular and hematologic side effects are dose-limiting because they may not allow for the administration of the dose required for maximum efficacy, limiting the possibility of achieving maximum therapeutic benefit (Humbert, M. et al., 2021). In contrast, the binding agents provided herein do not induce hematological effects in non-human primates, suggesting that these agents may have a broader therapeutic window than sotatercept.

[0102] In some embodiments, the binding agents of the present disclosure comprise homodimers, i.e., dimers of polypeptides having the sequence set forth in any one of SEQ ID NOS: 174-251, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or functionally equivalent variants thereof. In other embodiments, the binding agents comprise heterodimers, i.e., dimers of two different polypeptides, at least one of which has the sequence set forth in any one of SEQ ID NOS: 174-251, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or functionally equivalent variants thereof.

[0103] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising ActRIIB-ECD comprising the L33W mutation and a long peptide linker. In some embodiments, the peptide linker is 10 amino acids or at least 10 amino acids in length. In some embodiments, the peptide linker is 14 amino acids or at least 14 amino acids in length. In some embodiments, the peptide linker is 19 amino acids or at least 19 amino acids in length. In some embodiments, the peptide linker is 39 amino acids or at least 39 amino acids in length. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 211 and 230-234, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to this amino acid sequence, or a functionally equivalent variant thereof.

[0104] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO:211, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO:230, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO:231, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 232, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 233, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 234, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof.

[0105] Activin receptor type IIB ectodomain variant As used herein, the term "activin receptor type IIB ectodomain variant" or "ActRIIB-ECD variant" refers to a polypeptide comprising a soluble extracellular portion of the single-pass transmembrane receptor ActRIIB having at least one amino acid substitution relative to wild-type extracellular ActRIIB. The sequence of wild-type human ActRIIB-ECD is set forth in SEQ ID NO: 2 (Table 1). Unless otherwise specified, the indicated positions of amino acid substitutions are numbered according to the amino acid sequence of SEQ ID NO: 2. For purposes of this disclosure, "human wild-type ActRIIB-ECD" refers to SEQ ID NO: 2.

[0106] In some embodiments, the ActRIIB-ECD variant comprises one or more amino acid substitutions at a position selected from L14, G27, L33, L55, and T69. In some embodiments, the ActRIIB-ECD variant polypeptide comprises one or more amino acid substitutions selected from L14E, L14H, L14S, L14N, L14Q, L14D, G27E, G27D, G27N, G27Q, G27K, G27T, G27M, L33R, L33Y, L33F, L33Q, L33W, L33E, L33K, L33M, L55Y, L55Q, L55M, L55I, L69H, L69Q, L69E, L69R, L69Y, and L69W. In some embodiments, the ActRIIB-ECD variant comprises one or more amino acid substitutions selected from L33R, L33Y, L33F, L33Q, L33W, L33E, L33K, and L33M. In some embodiments, the ActRIIB ECD variant comprises an amino acid substitution of L33Y. In some embodiments, the ActRIIB ECD variant comprises an amino acid substitution of L33W. Other amino acid substitutions in ActRIIB-ECD are known in the art (e.g., WO2021 / 158675, WO2022 / 150590, WO2021 / 158675, WO2022 / 072882, WO2021 / 189019, and WO2021 / 189010, each of which is incorporated by reference herein). These additional mutations, in combination with the linkers described herein, can be incorporated into the binding agents described herein to alter the ligand binding properties of ActRIIB-ECD.

[0107] In some embodiments, the ActRIIB-ECD variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4-62. In some embodiments, the ActRIIB-ECD variant comprises at least 85% (e.g., at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more) amino acid sequence identity to the sequence of wild-type human ActRIIB-ECD. In some embodiments, the ActRIIB-ECD variant may have at least 85% (e.g., at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more) amino acid sequence identity to the sequence set forth in SEQ ID NO: 2.

[0108] In some embodiments, the amino acid sequence of the ActRIIB-ECD variant comprises at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 4-62.

[0109] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 4-9. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and comprises or consists of an amino acid sequence selected from SEQ ID NOs: 4-9. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27E and comprises or consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27N and comprises or consists of the amino acid sequence of SEQ ID NO: 5. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27Q and comprises or consists of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27K and comprises or consists of the amino acid sequence of SEQ ID NO: 7. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27T and comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27M and comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0110] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 10-18. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and comprises or consists of an amino acid sequence selected from SEQ ID NOs: 10-18. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33F and comprises or consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Q and comprises or consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33H and comprises or consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33R and comprises or consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33E and comprises or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33K and comprises or consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33M and comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0111] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 19-24. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and comprises or consists of the amino acid sequence of SEQ ID NO: 19-24. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69H and comprises or consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69Q and comprises or consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69E and comprises or consists of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69R and comprises or consists of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69Y and comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69W and comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0112] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and position T69 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 25-31. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and position T69 and comprises or consists of SEQ ID NOs: 25-31. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position T69R and comprises or consists of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position T69Y and comprises or consists of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position T69W and comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position T69H and comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position T69Q and comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position T69E and comprises or consists of the amino acid sequence of SEQ ID NO: 30. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33F and position T69Q and comprises or consists of the amino acid sequence of SEQ ID NO: 31.

[0113] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and position L33 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 32-48. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and position L33 and comprises or consists of an amino acid sequence of SEQ ID NO: 32-48. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27E and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27D and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27N and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27Q and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 35. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27K and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 36. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27T and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27M and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 38. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27D and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 39. In some embodiments, the ActRIIB-ECD variant comprises amino acid substitutions at positions G27E and L33Y and comprises or consists of the amino acid sequence of SEQ ID NO:40.In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27N and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27Q and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 42. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27D and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 43. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27E and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27N and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 45. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27Q and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27T and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 47. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27M and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 48.

[0114] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14 and position L33 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 49-58. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14 and position L33 and comprises or consists of an amino acid sequence of SEQ ID NO: 49. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14D and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 50. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14N and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 51. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14E and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 52. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14H and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14S and position L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14E and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 55. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14D and position L33W and comprises or consists of the amino acid sequence of SEQ ID NO: 56. In some embodiments, the ActRIIB-ECD variant comprises amino acid substitutions at positions L14N and L33W and comprises or consists of the amino acid sequence of SEQ ID NO:57.In some embodiments, the ActRIIB-ECD variant comprises amino acid substitutions at positions L14Q and L33W and comprises or consists of the amino acid sequence of SEQ ID NO:58.

[0115] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and position L55 and comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 59-62. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position L55Y and comprises or consists of the amino acid sequence of SEQ ID NO: 59. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position L55Q and comprises or consists of the amino acid sequence of SEQ ID NO: 60. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and position L55M and comprises or consists of the amino acid sequence of SEQ ID NO: 61. In some embodiments, the ActRIIB-ECD variant comprises amino acid substitutions at position L33Y and position L55I and comprises or consists of the amino acid sequence of SEQ ID NO:62.

[0116] An exemplary ActRIIB ECD is shown in Table 1. Amino acid substitutions are shown in bold, expanded text.

[0117] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0118] In some embodiments, an ActRIIB-ECD variant of the present disclosure further comprises an extension of up to 5 amino acids at the N-terminus. In some embodiments, an ActRIIB-ECD variant of the present disclosure further comprises a 5-amino acid extension at the N-terminus, e.g., GRGEA (SEQ ID NO: 63). In some embodiments, an ActRIIB-ECD variant of the present disclosure further comprises a 4-amino acid, 3-amino acid, 2-amino acid, or 1-amino acid extension at the N-terminus, e.g., but not limited to, RGEA, GEA, EA, or A. In some embodiments, an ActRIIB-ECD variant of the present disclosure further comprises a 3-amino acid extension at the C-terminus. In some embodiments, an ActRIIB-ECD variant of the present disclosure further comprises a 3-amino acid extension at the C-terminus, e.g., APT.

[0119] Exemplary ActRIIB ECDs with N- and C-terminal extensions are shown in Table 2. The amino acids of the extensions are shown in bold italic text. In some embodiments, any one of SEQ ID NOs: 4-62 can further comprise an N- or C-terminal extension.

[0120] [Table 2]

[0121] The ActRIIB-ECD variants disclosed herein are designed to maximize therapeutic efficacy in specific disease indications while minimizing adverse effects, specifically, to prevent or reduce inhibition of endogenous BMP-9 and / or BMP-10 signaling while maintaining and / or increasing neutralizing potency against other TGFβ superfamily ligands, such as activin A, activin B, GDF-8, and / or GDF-11. The ActRIIB-ECD variants of the present disclosure exhibit (1) similar or improved binding to activin A, activin B, GDF-8, and / or GDF-11 compared to wild-type ActRIIB-ECD (enabling them to compete with endogenous receptors for ligand binding and reduce or inhibit endogenous receptor signaling), and (2) reduced binding to BMP-9 compared to wild-type ActRIIB-ECD (avoiding the toxicity associated with inhibition of BMP-9 signaling), and optionally (3) similar or reduced binding to or inhibition of BMP-10 (avoiding the toxicity associated with inhibition of BMP-10 signaling). These variants can be used to treat a wide range of diseases and conditions in which activin receptor signaling is elevated, such as pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or hybrid PH), metabolic and cardiometabolic disorders (e.g., obesity, type 1 diabetes, type 2 diabetes, prediabetes, heart failure), bone diseases (e.g., diseases or conditions involving bone damage), muscle diseases, fibrosis, and low red blood cell levels (e.g., anemia, blood loss), as further described herein. The variants may result in, for example, but not limited to, a reduction in the symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or hybrid PH), a reduction in bone resorption or osteoclast activity, an increase in bone formation or bone mineral density, an increase in muscle mass or strength, a reduction in fibrosis (e.g., a decrease in fibrosis or a slowing or halting of the progression of fibrosis), and / or an increase in red blood cell levels (e.g., an increase in hemoglobin levels, hematocrit, or red blood cell count, e.g., increased red blood cell production), as further described herein.

[0122] In some embodiments, the ActRIIB-ECD variants of the present disclosure bind to one or more ligands selected from activin A, activin B, GDF-8, and GDF-11, and inhibit signaling through the receptors of the one or more ligands, respectively, without substantially binding to BMP-9 or BMP-10 and / or inhibiting BMP-9 or BMP-10 signaling through their receptor(s).

[0123] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-9 signaling is reduced by about 5-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 signaling.

[0124] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-9 signaling is reduced by about 10-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 signaling.

[0125] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-9 signaling is reduced by about 100-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 signaling.

[0126] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-10 signaling is reduced by about 5-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-10 signaling.

[0127] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-10 signaling is reduced by about 10-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-10 signaling.

[0128] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-10 signaling is reduced by about 100-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-10 signaling.

[0129] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against one or more ligands selected from activin A, activin B, BMP-8, and GDF-11 is increased compared to or substantially the same as the inhibitory potency of human wild-type ActRIIB-ECD against the same one or more ligands.

[0130] In some embodiments, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against activin A and lower inhibitory potency against BMP-9 and / or BMP-10 compared to human wild-type ActRIIB-ECD.

[0131] In some embodiments, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against activin B and lower inhibitory potency against BMP-9 and / or BMP-10 compared to human wild-type ActRIIB-ECD.

[0132] In some embodiments, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against both activin A and activin B, and lower inhibitory potency against BMP-9 and / or BMP-10, compared to human wild-type ActRIIB-ECD.

[0133] In some embodiments, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against GDF-8 and lower inhibitory potency against BMP-9 and / or BMP-10 compared to human wild-type ActRIIB-ECD.

[0134] In some embodiments, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against GDF-11 and lower inhibitory potency against BMP-9 and / or BMP-10 compared to human wild-type ActRIIB-ECD.

[0135] In some embodiments, the ActRIIB-ECD variants of the present disclosure have reduced inhibitory potency against BMP-10 compared to human wild-type ActRIIB-ECD.

[0136] In some embodiments, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against both activin A and / or activin B, lower inhibitory potency against BMP-9, and lower inhibitory potency against BMP-10 compared to human wild-type ActRIIB-ECD.

[0137] In some embodiments, the ActRIIB-ECD variants of the present disclosure do not cause vascular complications in a subject. In some embodiments, the ActRIIB-ECD variants of the present disclosure do not increase vascular permeability or leakage in a subject.

[0138] As a result, according to the present disclosure, novel polypeptides are provided herein comprising activin receptor type IIB (ActRIIB) ectodomain (ECD) variants, which have one or more amino acid substitutions compared to the sequence of human wild-type ActRIIB-ECD and have tailored TGFβ superfamily ligand specificity aimed at preventing or reducing disruption of endogenous BMP-9 and / or BMP-10 signaling while maintaining and / or increasing the neutralization potency of other TGFβ superfamily ligands, such as activin A, activin B, GDF-8, and / or GDF-11, to maximize therapeutic efficacy while minimizing adverse effects.

[0139] Polypeptides containing ActRIIB ECD variants In some embodiments, the present disclosure provides a polypeptide comprising an ActRIIB ECD variant fused to an Fc domain monomer via a linker. In some embodiments, the polypeptide comprises, from N- to C-terminus, an ActRIIB ECD variant-peptide linker-Fc domain monomer. The polypeptide comprising the ActRIIB ECD can dimerize via cysteine ​​bonds between the Fc domain monomers to form a TGFβ superfamily ligand binding agent described herein.

[0140] Linker In some embodiments, the ActRIIB ECD variant described herein is fused to a heterologous domain via a linker. In some embodiments, the heterologous domain increases the stability of the polypeptide. In some embodiments, the heterologous domain is selected from the group consisting of an Fc domain monomer (e.g., a wild-type Fc domain monomer, an Fc domain monomer with one or more amino acid substitutions), an albumin-binding peptide, a fibronectin domain, or a human serum albumin domain.

[0141] As used herein, the terms "peptide linker" and "linker" are used interchangeably to refer to a short stretch of amino acids used to connect two functional domains together within a polypeptide chain. For example, in some embodiments of the polypeptides or binding agents of the present disclosure, the ActRIIB-ECD variant and the Fc domain monomer are linked together on the polypeptide chain via one or more peptide linkers. Peptide linkers can also be used to attach other domains, modules, or regions (such as half-life extending domains) to the polypeptides or binding agents of the present disclosure. As used herein, the term "long linker" refers to a linker that is at least 10 amino acids long (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids long). As used herein, the term "short linker" refers to a linker that is less than 10 amino acids long (i.e., 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long).

[0142] Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the linker can include multiple or repeating motifs of the motif GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 126), GGGS (SEQ ID NO: 125), GGGG (SEQ ID NO: 104), GGGGA (SEQ ID NO: 124), GGGGS (SEQ ID NO: 103), GGGGG (SEQ ID NO: 123), GGAG (SEQ ID NO: 122), GGSG (SEQ ID NO: 121), AGGG (SEQ ID NO: 120), or SGGG (SEQ ID NO: 110).

[0143] In some embodiments, the linker can comprise 2 to 12 amino acids containing a GA or GS motif, e.g., GA, GS, GAGA (SEQ ID NO: 137), GSGS (SEQ ID NO: 129), GAGAGA (SEQ ID NO: 130), GSGSGS (SEQ ID NO: 131), GAGAGAGA (SEQ ID NO: 132), GSGSGSGS (SEQ ID NO: 133), GAGAGAGAGA (SEQ ID NO: 134), GSGSGSGSGS (SEQ ID NO: 135), GAGAGAGAGAGA (SEQ ID NO: 136), and GSGSGSGSGSGS (SEQ ID NO: 138). In some embodiments, the linker can comprise 3 to 12 amino acids containing a GGA or GGS motif, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 139), GGSGGS (SEQ ID NO: 140), GGAGGAGGA (SEQ ID NO: 141), GGSGGSGGS (SEQ ID NO: 142), GGAGGAGGAGGA (SEQ ID NO: 143), and GGSGGSGGSGGS (SEQ ID NO: 144). In some embodiments, the linker can comprise 4 to 12 amino acids including the motifs GGAG (SEQ ID NO: 145), GGSG (SEQ ID NO: 146), GGAGGGAG (SEQ ID NO: 147), GGSGGGSG (SEQ ID NO: 148), GGAGGGAGGGAG (SEQ ID NO: 149), and GGSGGGSGGGSG (SEQ ID NO: 150). In some embodiments, the linker can include the motifs GGGGA (SEQ ID NO: 124) or GGGGS (SEQ ID NO: 103), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 151) and GGGGSGGGGSGGGGGS (SEQ ID NO: 93). In some embodiments, the amino acid linker between the ActRIIB-ECD variant and the heterologous domain (e.g., an Fc domain monomer (e.g., a wild-type Fc domain monomer, an Fc domain monomer with one or more amino acid substitutions), an albumin-binding peptide, a fibronectin domain, or a human serum albumin domain) can be GGG, GGGA (SEQ ID NO: 126), GGGG (SEQ ID NO: 104), GGGAG (SEQ ID NO: 168), GGGAGG (SEQ ID NO: 169), or GGGAGGG (SEQ ID NO: 170).

[0144] When a linker is used, the linker is generally of sufficient length and sequence to ensure that each of the domains can retain their differential binding specificity and / or function independently of one another. In some embodiments, a peptide linker is selected that does not further promote any secondary structure. This linking of the domains to one another can be achieved by genetic engineering, for example, as described herein. Methods for preparing fused, operably linked polypeptide constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0145] In some embodiments, the linker comprises various permutations of amino acid sequences containing Gly and Ser. In some embodiments, the linker is a glycine- and serine-rich linker. In some embodiments, the linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, or 2-3 glycine residues) or glycine and proline residues, for example, a single threonine / serine and glycine sequence, a repeating threonine / serine and / or glycine sequence, for example, a singlet or repeat of GGG, GGGG (SEQ ID NO: 104), GGGS (SEQ ID NO: 125), TGGGG (SEQ ID NO: 108), SGGGG (SEQ ID NO: 109), TGGG (SEQ ID NO: 107), or SGGG (SEQ ID NO: 110). Other near-neutral amino acids, such as, but not limited to, Thr, Asn, Pro, and Ala, can also be used in the linker sequence.

[0146] In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is more than 10 amino acids in length. In some embodiments, the linker is at least 10, 11, 12, 13, 14, 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, or 50 amino acids in length. In some embodiments, the linker is less than 40, 35, 30, 25, 22, or 20 amino acids. In some embodiments, the linker is 10 to 50, 10 to 40, 10 to 30, 10 to 25, 10 to 21, 10 to 15, 10 to 14, 12 to 14, 15 to 25, 17 to 22, 20, or 21 amino acids in length. In some embodiments, the linker is 14 to 40, 14 to 39, 14 to 35, 14 to 30, 14 to 25, or 14 to 20 amino acids in length. In some embodiments, the linker is at least 10 amino acids in length. In some embodiments, the linker is at least 14 amino acids in length. In some embodiments, the linker is at least 19 amino acids in length. In some embodiments, the linker is at least 39 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 39 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 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, or 50 amino acids in length. In further embodiments, the linker has a length of at least 12, 14, 15, 20, 21, 25, 30, 35, 40, 45, or 50 amino acids.

[0147] In other embodiments, the linker is less than 10 amino acids in length. In some embodiments, the linker is 3 amino acids in length. In some embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 9 amino acids in length.

[0148] In some embodiments, the linker comprises SEQ ID NO: 98. In some embodiments, the linker comprises SEQ ID NO: 94. In some embodiments, the linker comprises SEQ ID NO: 89.

[0149] In some embodiments, the linker consists of SEQ ID NO: 98. In some embodiments, the linker consists of SEQ ID NO: 94. In some embodiments, the linker consists of SEQ ID NO: 89.

[0150] In some embodiments, the linker comprises or consists of a sequence set forth in any one of SEQ ID NOs: 68-170.

[0151] In some embodiments, the linker is a glycine-rich, often glycine / serine-rich, peptide consisting of up to 40 amino acids, or 1-40 amino acids, 2-39 amino acids, 3-39 amino acids, 3-14 amino acids, 3-19 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, or 15-25 amino acids. In some embodiments, the peptide linker contains only a relatively small number of amino acid residues, e.g., 39 or fewer amino acids, 19 or fewer amino acids, 14 or fewer amino acids, 5 or fewer amino acids, or 3 or fewer amino acids. In certain embodiments, Gly-rich linkers are used. In one embodiment, the peptide linker can consist of the single amino acid glycine (Gly). In another embodiment, the peptide linker comprises or consists of the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser, or a polymer thereof, i.e., (Gly4Ser)n, where n is an integer of 1 or greater, or n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0152] In some embodiments, the linker comprises the amino acid sequence GlyGlyGlyGlySer(GGGGS) (SEQ ID NO: 103), or repeats thereof (GGGGS)n, where n>2. In certain embodiments, n>3, or n=3-10. In some embodiments, n>4, or n=4-10. In some embodiments, n is 4 or less in a (GGGGS)n linker. In some embodiments, n=4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6. In some embodiments, n=3, 4, 5, 6, or 7. In some embodiments, linkers comprising a (GGGGS)n sequence also include an N-terminal threonine.

[0153] In some embodiments, the linker may include amino acids other than glycine, alanine, and serine, such as AAAL (SEQ ID NO: 152), AAAK (SEQ ID NO: 153), AAAR (SEQ ID NO: 154), EGKSSGSGSESKST (SEQ ID NO: 155), GSAGSAAGSGEF (SEQ ID NO: 156), AEAAAKEAAAKA (SEQ ID NO: 157), KESGSVSSEQLAQFRSLD (SEQ ID NO: 158), GENLYFQSGG (SEQ ID NO: 159), SACYCELS (SEQ ID NO: 160), RSIAT (SEQ ID NO: 161), RPACKIPNDLKQKVMNH (SEQ ID NO: 162), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 163), AAANSSIDLISVPVDSR (SEQ ID NO: 164), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 165). In some embodiments, the linker can include a motif, e.g., multiple or repeated motifs of EAAAK (SEQ ID NO: 166). In some embodiments, the linker can include a motif, e.g., multiple or repeated motifs of proline-rich sequences such as (XP)n (where X can be any amino acid (e.g., A, K, or E) and n is 1-5), and PAPAP (SEQ ID NO: 167).

[0154] The length of the peptide linker and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the linker can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0155] Non-limiting examples of linkers are shown in Table 3. It should be understood that the linker is not intended to be particularly limited and any suitable linker may be used, so long as the desired function of the polypeptide or binding agent (binding, neutralization, etc.) is provided.

[0156] [Table 3-1] [Table 3-2] [Table 3-3]

[0157] In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises one or more linkers having a sequence set forth in any one of SEQ ID NOs: 89, 94, or 98. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent comprises a 2-, 3-, 6-, 10-, 14-, 19-, or 39-amino acid long glycine-rich linker at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises a linker of SEQ ID NO: 89 at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises a linker of SEQ ID NO: 94 at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises a linker of SEQ ID NO: 98 at the C-terminus of the ActRIIB ECD variant polypeptide.

[0158] Fc domain monomers and Fc domains In some embodiments, the present disclosure provides a polypeptide comprising an ActRIIB-ECD variant described herein fused to an Fc domain monomer via a linker, hi some embodiments, the ActRIIB-ECD variant is fused C-terminally to the N-terminus of the Fc domain monomer via a linker.

[0159] As used herein, an "Fc domain monomer" describes a single-chain protein that, when associated with another Fc domain monomer, forms a functional Fc domain. The association of two Fc domain monomers creates one Fc domain. As used herein, an "Fc domain" describes the minimum region (in the context of a larger polypeptide) or the minimum protein fold (in the context of an isolated protein) that can bind to or be bound by an Fc receptor (FcR). When two Fc domain monomers associate, the resulting Fc domain has Fc receptor binding activity. Thus, the Fc domain is a dimeric structure that can bind to an Fc receptor. Unless otherwise specified, all references herein to a "variant Fc domain" should be understood to refer to a dimeric Fc domain, where each Fc domain monomer contains the referenced mutation.

[0160] As used herein, an Fc domain is understood to include a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains (CH2, CH3) of an IgG, and optionally, the flexible hinge N-terminal to these domains. Although the boundaries of an Fc domain monomer may vary, a human IgG heavy chain Fc domain monomer is usually defined as including residues C226 or P230 at its carboxy terminus. Unless otherwise specified, all references to amino acid positions in Fc domains and Fc domain monomers follow the EU index as set forth in Kabat (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). Fc can refer to this region in isolation or in the context of a polypeptide construct. It should be noted that polymorphism has been observed at many Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist between the sequences provided herein and those in the art. The Fc domain monomers comprised in the polypeptides or binding agents of the present disclosure may be IgG1, IgG2, IgG3, or IgG4 domains.

[0161] In exemplary embodiments, the polypeptides of the present disclosure comprise one or more constant regions of an antibody, e.g., the second constant domain (CH2) and / or the third constant domain (CH3) of an antibody heavy chain, or an Fc domain monomer of an antibody heavy chain. The antibody may be, for example, an IgG antibody, such as, but not limited to, an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the antibody is a human antibody, e.g., the Fc domain monomer comprises the constant region of a human IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the Fc domain monomer has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a human IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the Fc domain monomer comprises or consists of an Fc domain monomer of a human IgG1 antibody. In another specific embodiment, the Fc domain monomer comprises or consists of an Fc domain monomer of a human IgG2 antibody. In another specific embodiment, the Fc domain monomer comprises or consists of an Fc domain monomer of a human IgG4 antibody. Exemplary Fc domain sequences (including both the wild-type sequence, its polymorphs, and variant sequences) are provided in Table 4.

[0162] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0163] In some embodiments, the IgG1 sequences of the present disclosure further comprise a two amino acid extension at the N-terminus, eg, DK.

[0164] Generally, the ActRIIB-ECD polypeptide is configured such that the Fc domain monomer is linked at its N-terminus to the C-terminus of the ActRIIB-ECD variant, and the orientation of the construct is a single chain of (ActRIIB-ECD variant)-(linker)-(Fc domain monomer) for each ActRIIB-ECD polypeptide, from N-terminus to C-terminus. However, the orientation of the construct is not particularly limited, and other orientations are also contemplated. For example, in some embodiments, the Fc domain monomer may be linked at its C-terminus to the N-terminus of the ActRIIB-ECD variant.

[0165] In exemplary embodiments, an Fc domain monomer allows for the covalent assembly of two or more polypeptide chains, e.g., via disulfide bonds between cysteine ​​residues. In this manner, the Fc domain monomer acts as a dimerization domain, allowing the assembly of two ActRIIB-ECD polypeptide chains to form a dimer. According to the present disclosure, such dimers generally comprise two polypeptides, each comprising an ActIIRB-ECD variant linked to an Fc domain monomer described herein, thereby forming a bivalent TGFβ superfamily ligand binder. Thus, the binders described herein comprise two ActIIRB-ECD variants, a linker domain, and an Fc domain.

[0166] An Fc domain monomer generally contains one or more cysteine ​​residues for cross-linking a first polypeptide and a second polypeptide in a homodimeric construct. For example, an Fc domain monomer may contain at least two cysteine ​​residues for forming disulfide bridges between two polypeptides, thereby forming a dimer. In some embodiments of the present technology, an Fc domain monomer comprises or consists of a sequence set forth in any one of SEQ ID NOs: 252-292, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, an Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 253. In certain embodiments, an Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 266. In certain embodiments, an Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 256. In a specific embodiment, the Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO:255.

[0167] In some embodiments, the present disclosure provides binding agents comprising a variant Fc domain, i.e., a non-naturally occurring Fc domain, e.g., an Fc domain that includes one or more non-naturally occurring amino acid residues, substitutions, additions, deletions, etc.

[0168] In some embodiments of the present technology, the Fc domain is a variant Fc domain that forms a variant Fc domain on a polypeptide or binding agent that has desirable properties, such as increased half-life, compared to a naturally occurring (wild-type) Fc sequence. As used herein, "variant Fc domain" refers to a non-naturally occurring Fc domain, e.g., an Fc domain that includes one or more non-naturally occurring amino acid residues, one or more amino acid substitutions relative to a wild-type human constant domain, or one or more amino acid deletions, additions, and / or modifications.

[0169] There are many known polymorphs for the IgG1 Fc domain, including the "DEL" polymorph and the "EEM" polymorph. The DEL polymorph contains the amino acids DEL at positions 356, 357, and 358, respectively (also referred to herein as "Fc-DL," e.g., SEQ ID NO: 253). The EEM polymorph contains the amino acids EEM at positions 356, 357, and 358, respectively (also referred to herein as "Fc-EM," e.g., SEQ ID NO: 252). Two binding agents that are otherwise identical except for the presence of a DEL or EEM Fc domain are expected to exhibit similar properties in terms of ligand binding and therapeutic efficacy. In some embodiments of the present technology, the Fc domain is a DEL Fc domain ("DL"). In some embodiments of the present technology, the Fc domain is an EEM Fc domain ("EM"). Other polymorphs may also be used, for example, the IgG1 polymorphs of SEQ ID NOs: 252 to 253 and 258 to 266, the IgG2 polymorphs of SEQ ID NOs: 267 to 276, the IgG3 polymorphs of SEQ ID NOs: 277 to 283, and the IgG4 polymorphs of SEQ ID NOs: 284 to 292.

[0170] In some embodiments, a variant Fc domain formed by two variant Fc domain monomers has altered binding characteristics to an Fc receptor, such as FcRn, compared to a comparable molecule (e.g., a protein having the same amino acid sequence except for having a wild-type Fc domain monomer). The serum half-life of a protein comprising an Fc domain can be increased by increasing the binding affinity of the Fc domain to FcRn. In one embodiment, the Fc domain variant has an enhanced serum half-life relative to a comparable molecule. In certain embodiments, the Fc domain variant comprises at least one amino acid substitution at one or more positions selected from the group consisting of M252Y, S254T, and T256 (referred to herein as "YTE"; e.g., SEQ ID NOs: 254 and 255). In another embodiment, the Fc domain variant comprises a Y at position 252 (e.g., SEQ ID NOs: 256 and 257, referred to herein as "Fc-Y"). In another embodiment, the Fc domain variant comprises a T at position 254. In another embodiment, the Fc domain variant comprises an E at position 256.

[0171] Thus, in some embodiments of the present technology, an ActRIIB-ECD polypeptide comprises a variant Fc domain monomer that forms an Fc domain with increased in vivo half-life relative to a comparable molecule, hi some such embodiments, the Fc domain monomer of the ActRIIB-ECD polypeptide comprises a substitution of at least one amino acid residue selected from the group consisting of residues 252, 254, and 256.

[0172] In some embodiments, the ActRIIB-ECD polypeptide comprises a variant Fc domain monomer comprising at least one amino acid substitution selected from the group consisting of M252Y, S254T, and T256E. In such embodiments, the variant Fc domain monomer may further comprise one or more additional amino acid substitution(s), such as, but not limited to, E356D and M358L.

[0173] In some embodiments, the ActRIIB-ECD polypeptide comprises a variant Fc domain monomer comprising the following amino acid substitutions: M252Y, S254T, and T256E (referred to herein as "FcYTE" or "YTE"). In some embodiments, the FcYTE domain monomer is the DEL polymorph (referred to herein as YTE-DL, e.g., SEQ ID NO: 255). In some embodiments, the FcYTE domain monomer is the EEM polymorph (referred to herein as YTE-EM, e.g., SEQ ID NO: 254).

[0174] In some embodiments, the ActRIIB-ECD polypeptide comprises a variant Fc domain monomer comprising the following amino acid substitution: M252Y (referred to herein as "FcY"). In some embodiments, the FcY domain monomer is in the DEL polymorph (referred to herein as Y-DL, e.g., SEQ ID NO: 256). In some embodiments, the FcY domain monomer is in the EEM polymorph (referred to herein as Y-EM, e.g., SEQ ID NO: 257).

[0175] In some embodiments, the ActRIIB-ECD polypeptide comprises an Fc domain monomer comprising a lysine residue (K) at the C-terminus.

[0176] In some embodiments, a variant Fc domain for use in an ActRIIB-ECD polypeptide of the present disclosure (e.g., an Fc domain formed by two variant Fc domain monomers) comprises one or more amino acid substitutions that reduce aggregation and / or increase stability and / or half-life of the ActRIIB-ECD polypeptide compared to a naturally occurring Fc sequence. In some embodiments, the Fc domain is selected to provide one or more effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation (complement-dependent cytotoxicity or CDC), opsonization, etc. In one embodiment, the variant Fc domain has enhanced binding to an Fc receptor compared to a comparable molecule. In a specific embodiment, the variant Fc domain has enhanced binding to the neonatal Fc receptor FcRn. In another embodiment, the variant Fc domain and / or polypeptide or binding agent containing the variant Fc domain has a binding affinity for FcRn that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold greater than that of a comparable molecule. The serum half-life of a protein comprising an Fc domain can be increased by increasing the binding affinity of the Fc domain monomer for FcRn. Thus, in one embodiment, a polypeptide or binding agent comprising a variant Fc domain has an enhanced serum half-life compared to a comparable molecule.

[0177] Examples of means for extending the serum half-life of the polypeptides and binding agents of the present disclosure include peptides, proteins, or protein domains fused to or otherwise attached to the polypeptides and binding agents. The group of peptides, proteins, or protein domains includes peptides that bind to other proteins with favorable pharmacokinetic profiles in the human body, such as serum albumin (see WO 2009 / 127691). An alternative concept for such half-life extending peptides includes peptides that bind to the neonatal Fc receptor (FcRn, see WO 2007 / 098420), which can also be used in the polypeptides and binding agents of the present disclosure. The concept of binding larger domains of proteins or complete proteins includes, for example, fusions of human serum albumin, variants or mutants of human serum albumin (WO2011 / 051489, WO2012 / 059486, WO2012 / 150319, WO2013 / 135896, WO2014 / 072481, WO2013 / 075066) or domains thereof, as well as fusions of immunoglobulin constant regions (Fc domains) and variants thereof, as described herein. Such variants of the Fc domain may be optimized / modified to enable desired dimer or multimer pairing, to eliminate Fc receptor binding (e.g., Fcg receptors), to enhance binding to FcRn, or for other reasons. Another concept known in the art for extending the half-life of small protein compounds in the human body is PEGylation of such compounds, such as the polypeptides or binding agents of the present disclosure.

[0178] In one embodiment, the disclosure provides a binding agent, wherein the Fc domain is selected from the group consisting of 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 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, 347, 251, 252, 254, 25 and comprising an unnatural amino acid residue at one or more positions selected from the group consisting of: 79, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. Optionally, the Fc domain may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, 6,737,056, PCT Patent Publication Nos. WO01 / 58957, WO02 / 06919, WO04 / 016). 750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925, and WO06 / 020114). In certain embodiments, the present disclosure provides an Fc variant protein composition, wherein the Fc domain is 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 235G, 235H, 235G ... 36E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243 L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 2631,263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 2 80A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 29 8H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325 I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328 F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F , 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W. Optionally, the Fc domain may include additional and / or alternative amino acid substitutions known to those skilled in the art (see, e.g., U.S. Patent Nos. 5,624,821, 6,277,375, 6,737,056, PCT Patent Publication Nos. WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, and WO05 / 040217).

[0179] Additional Domains It is envisioned that the ActRIIB-ECD polypeptides and / or binding agents of the present disclosure may have additional binding specificities or additional functions in addition to their function of binding to the target TGFβ superfamily ligand(s) as specified. In some embodiments of the present technology, the ActRIIB-ECD polypeptides or binding agents can be conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, and / or a diagnostic moiety. In some embodiments, the polypeptides may have additional functions, such as a fully functional Fc constant domain that mediates antibody-dependent cellular cytotoxicity, such as through the recruitment of effector cells such as NK cells, by providing a label (e.g., fluorescent), by providing a therapeutic agent such as a toxin or radionuclide, and / or by providing a means to enhance serum half-life.

[0180] In some embodiments, the ActRIIB-ECD polypeptide described herein comprises ActRIIB-ECD, a linker, an Fc domain monomer, and one or more additional domains. In some embodiments, the one or more additional domains are selected from a fibronectin domain and a human serum albumin domain. As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to membrane-spanning receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID No. P02751. In other embodiments, the fibronectin domain is an adnectin protein.

[0181] In some embodiments, the polypeptide or binding agent of the present disclosure comprises an ActRIIB-ECD variant fused to one or more fibronectin domains. Binding to a fibronectin domain can improve the pharmacokinetics of a protein pharmaceutical. A fibronectin domain is a high-molecular-weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to membrane-spanning receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments of the present invention, a fibronectin domain is attached to the N-terminus or C-terminus (e.g., the C-terminus) of an ActRIIB-ECD variant described herein (e.g., an ActRIIB-ECD variant having an amino acid sequence set forth in any one of SEQ ID NOS: 4-62), thereby increasing the serum half-life of the ActRIIB-ECD variant. The fibronectin domain can be attached to the N-terminus or C-terminus of an ActRIIB-ECD variant, or the polypeptide, or the binding agent thereof, directly or through a linker. In some embodiments, the polypeptide or binding agent of the present disclosure can be fused to the N- or C-terminus of a fibronectin domain, for example, through conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the ActRIIB-ECD variant and the fibronectin domain. Without being bound by theory, it is expected that in some embodiments, the inclusion of a fibronectin domain in the ActRIIB-ECD variants described herein can result in long-term retention of the therapeutic protein through binding to integrins and extracellular matrix components such as collagen and fibrin.

[0182] By way of example, fibronectin domains that can be used in the methods, compositions, and polypeptides of the present disclosure are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt number P02751. In another embodiment, the fibronectin domain is an Adnectin protein.

[0183] As used herein, the term "human serum albumin" refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood. It constitutes approximately half of the serum proteins. In some embodiments, human serum albumin has the sequence of UniProt number P02768.

[0184] In some embodiments, the ActRIIB variants, polypeptides, or binding agents described herein can be fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of protein pharmaceuticals. Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and constitutes approximately half of serum proteins. It is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure necessary for proper distribution of body fluids between blood vessels and body tissues. In some embodiments, the serum albumin is human serum albumin. In some embodiments, human serum albumin is conjugated to the N-terminus or C-terminus (e.g., C-terminus) of an ActRIIB-ECD variant described herein (e.g., an ActRIIB-ECD variant having an amino acid sequence set forth in any one of SEQ ID NOs: 4-62) to increase the serum half-life of the ActRIIB-ECD variant. Human serum albumin can be conjugated directly or through a linker to the N-terminus or C-terminus of the ActRIIB-ECD variant.

[0185] As an example, serum albumin that can be used in the polypeptides and methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt number P02768. In some embodiments, the polypeptide or binding agent of the present disclosure can be fused to the N-terminus or C-terminus of human serum albumin, for example, through conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the ActRIIB-ECD variant and human serum albumin. Without being bound by theory, it is expected that in some embodiments, the inclusion of human serum albumin in the ActRIIB-ECD variants described herein can result in long-term retention of the therapeutic protein.

[0186] In some embodiments, a polypeptide or binding agent of the present disclosure further comprises a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization, an albumin-binding peptide, a fibronectin domain, or human serum albumin), which may be fused to the N-terminus or C-terminus (e.g., the C-terminus) of an ActRIIB-ECD variant, polypeptide, or binding agent via a linker or other covalent bond. A polypeptide comprising an ActRIIB-ECD variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or heterodimer) through interaction between two Fc domain monomers, which combine to form the Fc domain in the dimer. Further, in some embodiments, a polypeptide or binding agent described herein has a serum half-life of at least 7 days in humans.

[0187] Exemplary TGFβ Superfamily Binding Agents The overall structures of exemplary binding agents described herein are shown in Table 5. The amino acid sequence of each binding agent is shown in Table 6.

[0188] [Table 5-1] Table 5-2 Table 5-3 Table 5-4 Table 5-5

[0189] Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9

[0190] In some embodiments, the binding agent comprises, from N-terminus to C-terminus, an ActRIIB ECD, a peptide linker, and an Fc domain. In some embodiments, the ActRIIB ECD comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions are at a position selected from L14, G27, L33, L55, and L69, where amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the substitution at position L14 is selected from L14E, L14H, L14S, L14N, L14Q, and L14D. In some embodiments, the amino acid substitution at position G27 is selected from G27E, G27D, G27N, G27Q, G27Q, G27K, G27T, and G27M. In some embodiments, the amino acid substitution at position L33 is selected from L33R, L33Y, L33F, L33Q, L33W, L33E, L33K, and L33M. In some embodiments, the amino acid substitution at position L55 is selected from L55Y, L55Q, L55M, and L55I. In some embodiments, the substitution at position T69 is selected from T69H, T69Q, T69E, T69R, T69Y, and T69W.

[0191] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a G27E amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO:4, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 174 (P739). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 174 (P739).

[0192] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33R amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 15, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 175 (P750). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 175 (P750).

[0193] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33Y amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 176 (P751). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 176 (P751).

[0194] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a T69H amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 19, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 177 (P753). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 177 (P753).

[0195] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a T69Q amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 20, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 178 (P754). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 178 (P754).

[0196] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a G27E amino acid substitution, a 3 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO:4, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 179 (P765). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 179 (P765).

[0197] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33Y amino acid substitution, a 3 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 180 (P777). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 180 (P777).

[0198] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a T69H amino acid substitution, a 3 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 19, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 181 (P779). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 181 (P779).

[0199] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a T69Q amino acid substitution, a 3 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 20, a 3 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 182 (P780). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 182 (P780).

[0200] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and T69H, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 28, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 183 (P1171). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 183 (P1171). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 184 (P1172). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 184 (P1172).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 185 (P1173). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 185 (P1173).

[0201] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and T69Q, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 29, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 186 (P1174). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 186 (P1174). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 187 (P1175). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 187 (P1175).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 188 (P1176). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 188 (P1176).

[0202] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising L33Y and T69E amino acid substitutions, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 30, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 189 (P1177). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 189 (P1177). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 190 (P1178). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 190 (P1178).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 191 (P1179). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 191 (P1179).

[0203] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and G27D, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 33, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 192 (P1180). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 192 (P1180). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 193 (P1181). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 193 (P1181).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 194 (P1182). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 194 (P1182).

[0204] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising L33Y and G27E amino acid substitutions, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 32, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 195 (P1183). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 195 (P1183). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 196 (P1184). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 196 (P1184).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 197 (P1185). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 197 (P1185).

[0205] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33F and T69Q, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 31, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 198 (P1186). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 198 (P1186). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 199 (P1187). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 199 (P1187).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 200 (P1188). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 200 (P1188).

[0206] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a T69E amino acid substitution, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 21, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 (P1201). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 201 (P1201). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202 (P1202). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 202 (P1202).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 203 (P1203). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 203 (P1203).

[0207] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising an L33F amino acid substitution, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 10, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204 (P1204). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 204 (P1204). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 205 (P1205). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 205 (P1205).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 206 (P1206). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 206 (P1206).

[0208] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33Q amino acid substitution, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, a peptide linker 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N- to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 11, a peptide linker of 3 aa, 10 aa, or 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 207 (P1207). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 207 (P1207). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 208 (P1208). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 208 (P1208).In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 209 (P1209). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 209 (P1209).

[0209] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33Y amino acid substitution, a 10 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, a 10 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a 10 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 210 (P1210). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 210 (P1210).

[0210] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33W amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 211 (P1229). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 211 (P1229).

[0211] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33E amino acid substitution, a 14 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, a 14 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 16, a 14 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 212 (P1230). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 212 (P1230).

[0212] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33K amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 17, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 213 (P1231). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 213 (P1231).

[0213] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33M amino acid substitution, a 14 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, a 14 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 18, a 14 aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 214 (P1232). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 214 (P1232).

[0214] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and G27N, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 34, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 215 (P1235). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 215 (P1235).

[0215] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and G27Q, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 35, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 216 (P1236). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 216 (P1236).

[0216] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and G27K, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 36, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 217 (P1237). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 217 (P1237).

[0217] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising L33Y and G27Y amino acid substitutions, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 37, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 218 (P1238). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 218 (P1238).

[0218] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and G27M, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 38, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 219 (P1239). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 219 (P1239).

[0219] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and T69R, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 25, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 220 (P1240). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 220 (P1240).

[0220] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising L33Y and T69Y amino acid substitutions, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 26, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 221 (P1241). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 221 (P1241).

[0221] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and T69W, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 27, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 222 (P1242). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 222 (P1242).

[0222] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and L14E, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 52, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 223 (P1269). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 223 (P1269).

[0223] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and L14H, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 53, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 224 (P1270). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 224 (P1270).

[0224] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and L14S, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 54, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 54, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 225 (P1271). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 225 (P1271).

[0225] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising L33Y and L55Y amino acid substitutions, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 59, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 59, a 14-aa peptide linker in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 226 (P1272). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 226 (P1272).

[0226] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and L55Q, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 60, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 60, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 227 (P1273). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 227 (P1273).

[0227] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and L55M, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 61, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 228 (P1274). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 228 (P1274). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L33Y and L55I, a peptide linker 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 62, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 62, a peptide linker 14 aa in length, and an IgG1-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 229 (P1275). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 229 (P1275).

[0228] In some embodiments, the TGFβ superfamily ligand-binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33W amino acid substitution, a 14-aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand-binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, a 14-aa peptide linker in length, and an IgG1 Y-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand-binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a 14-aa peptide linker in length, and an IgG1 Y-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 230 (P1371). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 230 (P1371).

[0229] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the L33W amino acid substitution, a 14 aa peptide linker in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, a 14 aa peptide linker in length, and an IgG1 YTE-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a 14 aa peptide linker in length, and an IgG1 YTE-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 231 (P1372). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 231 (P1372).

[0230] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33W amino acid substitution, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 232 (P1373). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 232 (P1373).

[0231] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an L33W amino acid substitution, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, a peptide linker 19 aa in length, and an IgG1 Y-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a peptide linker 19 aa in length, and an IgG1 Y-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 233 (P1374). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 233 (P1374).

[0232] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the L33W amino acid substitution, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, a peptide linker 19 aa in length, and an IgG1 YTE-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a peptide linker 19 aa in length, and an IgG1 YTE-DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 234 (P1375). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 234 (P1375).

[0233] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27D and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 43, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 235 (P1385). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 235 (P1385).

[0234] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27E and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 44, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 236 (P1386). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 236 (P1386).

[0235] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27N and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 45, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 237 (P1387). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 237 (P1387).

[0236] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27Q and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 46, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 238 (P1388). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 238 (P1388).

[0237] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27T and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 47, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 239 (P1389). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 239 (P1389).

[0238] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27M and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 48, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 48, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 240 (P1390). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 240 (P1390).

[0239] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27D and L33Y, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 39, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 241 (P1391). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 241 (P1391).

[0240] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising G27E and L33Y amino acid substitutions, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 40, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 242 (P1392). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 242 (P1392).

[0241] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising G27N and L33Y amino acid substitutions, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 41, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 243 (P1395). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 243 (P1395).

[0242] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions G27Q and L33Y, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 42, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 244 (P1396). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 244 (P1396).

[0243] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14E and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 55, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 55, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 245 (P1406). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 245 (P1406).

[0244] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14D and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 56, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 56, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 246 (P1407). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 246 (P1407).

[0245] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14N and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 57, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 57, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 247 (P1408). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 247 (P1408).

[0246] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14Q and L33W, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 58, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 58, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 248 (P1409). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 248 (P1409).

[0247] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14Q and L33Y, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 49, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 249 (P1410). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 249 (P1410).

[0248] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14D and L33Y, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 50, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 50, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 250 (P1411). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 250 (P1411).

[0249] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising the amino acid substitutions L14N and L33Y, a peptide linker 19 aa in length, and an IgG1 Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 51, a peptide linker 19 aa in length, and an IgG1 DL Fc domain monomer. In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 251 (P1412). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 251 (P1412).

[0250] In some embodiments, the polypeptides or binding agents of the present disclosure are "isolated" or "substantially pure." When used to describe a polypeptide or binding agent disclosed herein, "isolated" or "substantially pure" refers to a polypeptide or binding agent that has been identified, separated, and / or recovered from components of its production environment. Preferably, the polypeptide or binding agent is free or substantially free from association with all other components from its production environment. Contaminating components of its production environment, such as those arising from recombinantly transfected cells, are typically substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The polypeptide or binding agent may, for example, constitute at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that an isolated protein may constitute 5% to 99.9% by weight of the total protein content, depending on the context. The polypeptide or binding agent may be produced at significantly higher concentrations through the use of inducible promoters or high-expression promoters, such that it is produced at increased concentration levels. This definition includes production of polypeptides or binding agents in a wide variety of organisms and / or host cells known in the art. In preferred embodiments, the polypeptide or binding agent will be purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. However, typically, an isolated polypeptide or binding agent will be prepared by at least one purification step, such as, but not limited to, affinity and / or ion exchange chromatography, e.g., binding to a Protein A column.

[0251] In some embodiments, the polypeptides and binding agents of the present disclosure are characterized by, for example, one or more of: particularly high affinity for one or more of activin A, activin B, GDF-8, and GDF-11; high neutralizing potency (low IC50 values) for one or more of activin A, activin B, GDF-8, and GDF-11; particularly low or undetectable affinity for BMP-9 and / or BMP-10; low or undetectable neutralizing potency (high IC50 values) for BMP-9 and / or BMP-10; high thermal stability; high plasma stability; long or extended half-life; low turbidity; high protein homogeneity; and / or high manufacturability.

[0252] The biological activity of a polypeptide or binding agent of the present disclosure, or a pharmaceutical composition thereof, can be determined, for example, by cell neutralization assays, binding assays, competition assays, etc. As used herein, "efficacy" or "in vivo efficacy" refers to the response to therapy using a polypeptide or binding agent or pharmaceutical composition of the present disclosure. The success of therapy or in vivo efficacy using a polypeptide or binding agent or pharmaceutical composition of the present disclosure refers to the effectiveness of the polypeptide or binding agent or composition for its intended purpose, e.g., the ability of the polypeptide or binding agent or composition to cause the desired effect, i.e., the treatment, amelioration, or prevention of a TGFβ superfamily-related disease or disorder as defined herein. In vivo efficacy may be monitored by standard methods established for each disease entity. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used.

[0253] Another major challenge in the development of drugs, such as the pharmaceutical compositions of the present disclosure, is the predictable modulation of pharmacokinetic properties. To this end, the pharmacokinetic profile of a drug candidate can be established, i.e., a profile of pharmacokinetic parameters that affect the ability of a particular drug to treat a given condition. Pharmacokinetic parameters of a drug that affect the ability of a drug to treat a particular disease entity include, but are not limited to, half-life, volume of distribution, first-pass hepatic metabolism, and degree of serum binding. The efficacy of a given drug can be affected by each of the above parameters.

[0254] Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rate, onset of more, and / or Cmax for a given amount of administered drug. "Bioavailability" refers to the amount of drug in the blood compartment. "Lag time" refers to the time delay between administration of a drug and its detection and measurability in the blood or plasma. "Tmax" is the time after which the maximum blood concentration of the drug is reached, and "Cmax" is the maximum obtainable blood concentration for a given drug. The time to reach the blood or tissue concentration of a drug required for its biological effect is influenced by all parameters.

[0255] In some embodiments, a polypeptide or binding agent of the disclosure has a half-life of about 3 days or more, about 5 days or more, about 1 week or more, about 2 weeks or more, about 3 weeks or more, about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, or about 2 months or more.

[0256] In some embodiments, a polypeptide or binding agent of the present disclosure may exhibit favorable thermal stability with an aggregation temperature of about 45°C or higher, about 45 to about 50°C, about 52 to about 54°C, about 56 to about 60°C, or about 60°C or higher. Thermal stability parameters can be determined in terms of polypeptide aggregation temperature as follows: A protein solution at a test concentration (e.g., 100 μg / ml, 250 μg / ml) is transferred to a single-use cuvette and placed in a dynamic light scattering (DLS) device. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min, and a measured radius is obtained at constant speed. The increase in radius, indicative of protein melting and aggregation, is used to calculate the aggregation temperature of the polypeptide. Other methods known in the art may also be used.

[0257] In one embodiment, a polypeptide or binding agent according to the present disclosure is stable at 2-8°C for at least 1 month, 2 months, or 3 months. In one embodiment, a polypeptide or binding agent according to the present disclosure is stable at 25-40°C for at least 4 weeks. In one embodiment, a polypeptide or binding agent according to the present disclosure is stable after undergoing three freeze / thaw cycles. In one embodiment, a polypeptide or binding agent according to the present disclosure is stable at -20°C for 1 month, 2 months, 3 months, or more.

[0258] Alternatively, temperature melting curves can be determined by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of a polypeptide or binding agent. These experiments can be performed using a MicroCal LLC (Northampton, Mass., USA) VP-DSC device. The energy uptake of a sample containing the polypeptide or binding agent, compared to a sample containing only the formulation buffer, is recorded from 20°C to 90°C. For each melting curve, the overall sample temperature is increased stepwise. At each temperature, the T energy uptake of the sample and formulation buffer reference is recorded. The difference in energy uptake Cp (kcal / mol / °C) of the sample minus the reference is plotted against each temperature. The melting temperature is defined as the temperature at the first maximum in energy uptake.

[0259] In further embodiments, the polypeptide or binding agent according to the present disclosure is stable at acidic pH. The more tolerant the polypeptide or binding agent is at non-physiological pH, such as pH 5.5 (e.g., the pH required for performing cation exchange chromatography), the higher the recovery of the polypeptide or binding agent eluted from the ion exchange column relative to the total amount of loaded protein. The recovery of the polypeptide or binding agent from an ion (e.g., cation) exchange column at pH 5.5 can be 50% or more, 60% or more, 65% or more, 70% or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 90% or more, 95% or more, or 99% or more.

[0260] Amino acid sequence modification Modifications to the amino acid sequences of the polypeptides and binding agents described herein are contemplated. For example, it may be desirable to improve the binding affinity, effector function, half-life, and / or other biological properties of the polypeptides or binding agents. Amino acid sequence modifications / variants of the polypeptides and binding agents are generally prepared by introducing appropriate nucleotide changes into the encoding nucleic acid or by peptide synthesis. All of the amino acid sequence modifications described below must result in a polypeptide or binding agent that still retains the desired biological activity of the unmodified parent molecule (e.g., binding to one or more of activin A, activin, GDF-8, and GDF-11 without substantially binding to BMP-9 and BMP-10).

[0261] As used herein, the term "functionally equivalent" refers to a modified sequence that has the same or substantially the same biological activity or function as the original sequence from which it is derived, e.g., no significant changes in physiological, chemical, physicochemical, or functional properties compared to the original sequence. The term "substantially identical" refers to a sequence that is functionally equivalent to and has a high degree of sequence identity with the original or reference sequence. Generally, a substantially identical sequence is at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the original or reference sequence and has the same function. In some cases, when referring to nucleic acid sequences, a substantially identical sequence hybridizes to the original sequence under high stringency conditions, e.g., salt and temperature conditions substantially equivalent to 0.5X SSC to about 5X SSC and 65°C for both hybridization and washing. Generally, modified sequences that are substantially identical or functionally equivalent to the sequences provided in accordance with the present disclosure are intended to be encompassed.

[0262] Amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the polypeptide or binding agent. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processing of the polypeptide or binding agent, such as changing the number or location of glycosylation sites. In certain embodiments, one or more amino acids are altered to modify a glycosylation site.

[0263] For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or deleted from the polypeptide or binding agent. Preferably, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues up to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Insertional variants of the polypeptides or binding agents of the present disclosure include fusions to the N- or C-terminus of the polypeptide or binding agent of an enzyme, or to a polypeptide that increases the serum half-life of the polypeptide or binding agent.

[0264] Modifications of the polypeptides or binding agents described herein are also contemplated. Modifications encompassed by the present disclosure include those having alterations in the amino acid sequence of the polypeptide or binding agent. Modified polypeptides or binding agents include, for example, those having similar or improved binding affinity, affinity, ligand specificity, inhibitory potency, stability, manufacturability, half-life, and / or reduced aggregation compared to the polypeptides or binding agents disclosed herein.

[0265] One site of interest for substitutional mutagenesis includes the Fc domain monomers described herein above. Exemplary embodiments of modified polypeptides or binding agents of the present disclosure may include those with modified IgG1, IgG2, IgG3, or IgG4 constant regions or portions thereof. In one embodiment, the polypeptide or binding agent comprises an IgG1 constant region (modified or unmodified). In one embodiment, the polypeptide or binding agent comprises an IgG2 constant region (modified or unmodified). In one embodiment, the polypeptide or binding agent comprises an IgG3 constant region (modified or unmodified). In one embodiment, the polypeptide or binding agent comprises an IgG4 constant region (modified or unmodified).

[0266] Modifications encompassed by the present disclosure include modifications that may include insertions, deletions, or amino acid substitutions (conservative or non-conservative). These modifications may involve removing at least one amino acid residue in the amino acid sequence and inserting a different residue in its place. It should be understood that variations may occur in multiple regions of a polypeptide or binding agent, as long as the desired binding or biological activity is maintained.

[0267] Modifications and variants are known in the art to be capable of being generated by substitution mutagenesis and retaining the biological activity (i.e., functional equivalence) of the polypeptides of the present disclosure. These modifications or variants have at least one amino acid residue in the amino acid sequence removed and a different residue inserted in its place, e.g., one or more conservative amino acid substitutions. Generally, a conservative amino acid substitution is the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).

[0268] In general, the degree of similarity and identity between variant polypeptide chains is determined herein using the Blast2 sequence program (Tatusova, TA and Madden, TL, 1999) using default settings, i.e., the blastp program, the BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gap dropoff 50, expectation 10.0, word size 3), and an activation filter.

[0269] However, the level of identity may be determined over the entire length of a given sequence. Thus, percent identity indicates the amino acids that are identical and may occupy the same or similar positions compared to the original peptide. Percent similarity indicates the amino acids that are identical and that are substituted with conservative amino acid substitutions compared to the original peptide at the same or similar positions.

[0270] In some embodiments, modifications of the polypeptides or binding agents of the present disclosure therefore comprise an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the original sequence or a portion of the original sequence.

[0271] In some embodiments, the substitutions are conservative substitutions. However, any substitution (including non-conservative substitutions) is contemplated as long as the polypeptide or binding agent retains the ability to bind to and / or inhibit the desired TGFβ superfamily ligand without substantially binding to or inhibiting BMP-9 and / or BMP-10.

[0272] Generally, the nucleic acid sequence homology, similarity, or identity between a nucleotide sequence encoding a polypeptide or binding agent of the disclosure and the nucleotide sequences set forth herein is at least 60%, and more typically at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and increasingly greater homology or identity up to approximately 100%.

[0273] Production of nucleic acids and polypeptides and binding agents The present disclosure further provides polynucleotides encoding the ActRIIB-ECD polypeptides provided herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 293-296 or 298-375. In some embodiments, the polynucleotide comprises or consists of any one of SEQ ID NOs: 293-296 or 298-375. In some embodiments, the polypeptide encodes an amino acid sequence having 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 174-251. In some embodiments, the polypeptide encodes an amino acid sequence comprising or consisting of SEQ ID NOs: 174-251.

[0274] A polynucleotide is a biopolymer composed of nucleotide monomers covalently linked in a chain. DNA (e.g., cDNA) and RNA (e.g., mRNA) are examples of polynucleotides with different biological functions. Nucleotides are organic molecules that function as monomers or subunits of nucleic acid molecules such as DNA or RNA. A nucleic acid molecule or polynucleotide can be double-stranded or single-stranded, linear or circular. In some embodiments, the nucleic acid molecule or polynucleotide is contained in a vector. In some embodiments, the vector is contained in a host cell. The host cell is capable of expressing a polypeptide or binding agent, for example, after transformation or transfection with a vector or polynucleotide of the present disclosure. To that end, the polynucleotide or nucleic acid molecule is usually operably linked to a regulatory sequence.

[0275] Additionally, the present disclosure provides vectors comprising polynucleotides / nucleic acid molecules encoding the polypeptides or binding agents provided herein.

[0276] A vector is a nucleic acid molecule used as a vehicle for introducing (foreign) genetic material into cells. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Engineered vectors generally contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is generally a nucleotide sequence, typically a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may contain additional features beyond the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically intended for the expression of transgenes in target cells and generally contain regulatory sequences.

[0277] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0278] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader (signal sequence) is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at conventional restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0279] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term is primarily used for non-viral methods in eukaryotic cells. Transduction is often used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection can be performed using calcium phosphate, by electroporation, by squeezing the cells, or by mixing cationic lipids with the material to generate liposomes, which fuse with the cell membrane and store the cargo inside.

[0280] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and also into non-animal eukaryotic cells, including plant cells. Transformation is thus the genetic change of bacteria or non-animal eukaryotic cells resulting from the direct uptake and subsequent incorporation of exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane(s). Transformation can also be achieved by artificial means. For transformation to occur, the cells or bacteria must be in a competent state, which can occur as a time-limited response to environmental conditions such as starvation or cell density.

[0281] Additionally, the present disclosure provides host cells transformed or transfected with the polynucleotide / nucleic acid molecules or vectors of the technology.

[0282] As used herein, the terms "host cell" and "recipient cell" are intended to include any individual cell or cell culture that can be or is a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding a polypeptide or binding agent of the present disclosure, and / or the polypeptide or binding agent itself. Introduction of the respective substance into a cell is accomplished by transformation, transfection, etc. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Because certain modifications may occur in subsequent generations due to either natural, accidental, or deliberate mutation, or due to environmental influences, such progeny may not actually be completely identical to the parent cell (either morphologically or in genomic or total DNA complement), but still fall within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, e.g., mouse cells, rat cells, macaque cells, or human cells.

[0283] The polypeptides or binding agents of the present disclosure can be produced in bacteria. After expression, the polypeptides or binding agents can be isolated from the E. coli cell paste in a soluble fraction and purified, for example, through affinity chromatography and / or size exclusion. Final purification can be carried out, for example, similar to the process for purifying proteins expressed in CHO cells.

[0284] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptides or binding agents. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, Schizosaccharomyces pombe, Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402 226); Pichia pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous Several other genera, species, and strains of fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are commonly available and useful herein.

[0285] Suitable host cells for expressing the glycosylated polypeptides or binding agents of the present disclosure are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as viruses herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells.

[0286] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, sea anemone, and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins in plant cell culture are well known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Oven et al. (1992) Bio / Technology 10:790-794; Arsaenko et al. (1995) The Plant J 8:745-750; and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0287] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980); mouse Sertoli cells (TM4, Mather, 1980); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383: 44-68); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0288] In a further embodiment, the disclosure provides a process for the production of a polypeptide or binding agent, the process comprising culturing a host cell under conditions that allow expression of the polypeptide or binding agent, and recovering the produced polypeptide or binding agent from the culture.

[0289] As used herein, the term "culturing" refers to the in vitro maintenance, differentiation, growth, proliferation, and / or propagation of cells under suitable conditions in a culture medium. The term "expression" includes any step involved in the production of a polypeptide or binding agent of the disclosure, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0290] When using recombinant techniques, the polypeptide or binding agent can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide or binding agent is produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0291] Polypeptides or binding agents of the present disclosure prepared from host cells can be recovered or purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody recovered. When a polypeptide or binding agent of the present disclosure contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) can be useful for purification.

[0292] Affinity chromatography is a common purification technique. The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.

[0293] The polypeptides or binding agents disclosed herein may be made by a variety of methods well known to those skilled in the art, including recombinant DNA methods.

[0294] To express a polypeptide or binding agent, a nucleotide sequence capable of encoding a polypeptide chain described herein can be inserted into an expression vector, i.e., a vector containing elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements can include regulatory sequences, e.g., enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions. Such expression vectors can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo genetic recombination, etc.

[0295] A variety of expression vectors and host cell systems known to those skilled in the art may be used to express the polypeptide chains described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; and animal cell systems. For long-term production of recombinant proteins in mammalian systems, stable expression in mammalian cell lines may be used. For example, a nucleotide sequence capable of encoding any one of the polypeptide chains described herein may be transformed into a cell line using an expression vector that may contain a viral origin of replication and / or endogenous expression elements and a selectable or visible marker gene on the same or a separate vector. The present disclosure is not limited by the vector or host cell used. In certain embodiments disclosed herein, a nucleic acid capable of encoding a polypeptide chain described herein may be ligated into an expression vector. If the binding agent is composed of separate polypeptide chains (i.e., the first polypeptide and the second polypeptide are not identical), each of such polypeptide chains may be ligated into a separate vector or into the same vector. According to the present disclosure, the polypeptide chains of a binding agent may be encoded by a single vector or by separate vectors (e.g., a vector set). Cells are transformed with the desired vector or vector set.

[0296] Alternatively, the polypeptide chains can be expressed from an in vitro transcription system or a coupled in vitro transcription / translation system, respectively, or any such cell-free system.

[0297] Host cells containing the nucleotide sequences can be cultured under conditions for transcription of the corresponding RNA (such as mRNA) and / or expression and secretion of the polypeptide(s) from the cell culture. In exemplary embodiments, expression vectors containing nucleotide sequences capable of encoding the polypeptide chains described herein can be designed to contain a signal sequence that directs secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane.

[0298] Due to the inherent degeneracy of the genetic code, DNA sequences encoding identical, substantially identical, or functionally equivalent amino acid sequences may be produced and used. The nucleotide sequences of the present disclosure may be manipulated using methods generally known in the art to alter the nucleotide sequence for various purposes, including, but not limited to, modifying the cloning, processing, and / or expression of gene products. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to manipulate the nucleotide sequence. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variants, and the like. Codon-optimized nucleic acids encoding the polypeptide chains described herein are encompassed by the present disclosure.

[0299] In addition, a host cell line may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Different host cells (e.g., CHO, HeLa, MDCK, HEK293, and W138) with specific cellular and characteristic machinery for post-translational activity are available commercially and from the American Type Culture Collection (ATCC) and can be chosen to ensure the correct modification and processing of the expressed polypeptide.

[0300] Those skilled in the art will also readily recognize that nucleic acid and polypeptide sequences can be synthesized in whole or in part using chemical or enzymatic methods well known in the art. For example, peptide synthesis can be performed using various solid-phase techniques, or synthesis can be automated using machines such as the ABI 431A Peptide Synthesizer (PE Biosystems). If desired, the amino acid sequence can be modified during synthesis and / or combined with sequences from other proteins to produce variant proteins.

[0301] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising the polypeptides or TGFβ superfamily ligand binding agents disclosed herein. Pharmaceutical compositions generally comprise the polypeptides or binding agents disclosed herein and a pharmaceutically acceptable carrier.

[0302] Pharmaceutical compositions can be prepared as known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition, 2000). For example, the therapeutic compound and / or composition, together with one or more solid or liquid pharmaceutical carrier substances and / or additives (or auxiliary substances), and optionally in combination with other pharmaceutically active compounds having a therapeutic or prophylactic effect, can be brought into a suitable dosage form or formulation, which can then be used as a drug in human or veterinary medicine. Pharmaceutical formulations can also contain additives, many of which are known in the art, such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersing agents, preservatives, sweeteners, colorants, flavors, fragrances, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for modifying osmotic pressure, coating agents, or antioxidants.

[0303] The term "pharmaceutical composition" refers to a composition comprising at least one component including a polypeptide or binding agent described herein and a pharmaceutically acceptable carrier, diluent, adjuvant, excipient, or vehicle, such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, fragrances, antibacterial agents, antifungal agents, lubricants, and dispensing agents, depending on the mode of administration and the nature of the dosage form.

[0304] The term "pharmaceutically acceptable carrier" refers to any carrier, diluent, adjuvant, excipient, or solvent described herein or known in the art. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of absorption delaying agents, such as aluminum monostearate and gelatin. Non-limiting examples of suitable carriers, diluents, solvents, or vehicles include water, salt solution, phosphate buffered saline (PBS), gelatin, oils, alcohols, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Non-limiting examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Non-limiting examples of disintegrants include starch, alginic acid, and certain complex silicates. Non-limiting examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol.

[0305] The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with the cells of subjects, e.g., humans and animals, without undue toxicity, irritation, allergic response, and the like, and commensurate with a reasonable benefit / risk ratio.

[0306] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" is a term well known in the art and includes, but is not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Furthermore, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's oil, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antimicrobials, antioxidants, collating agents, inert gases, and the like, may also be present.

[0307] Pharmaceutically acceptable carriers may include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In one embodiment, the carrier is suitable for parenteral administration. The carrier may be suitable for intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. In other embodiments, the carrier is suitable for topical administration or administration by inhalation. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions provided herein is contemplated. Supplementary active compounds can also be incorporated into the compositions. For example, the pharmaceutical compositions provided herein may further comprise at least one additional therapeutic agent, as further described below.

[0308] The pharmaceutical compositions described herein may be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0309] In some embodiments, the pharmaceutical compositions provided herein can be administered orally, for example, in the form of pills, tablets, lacquered tablets, dragees, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example, in the form of suppositories.

[0310] In other embodiments, the pharmaceutical compositions provided herein can be administered parenterally, e.g., subcutaneously, intramuscularly, or intravenously, in the form of a solution for injection or infusion. Other suitable forms of administration are, for example, transdermal or topical administration in the form of an ointment, cream, tincture, spray, or transdermal therapeutic system, or inhalation administration in the form of a nasal spray or aerosol mixture, or, for example, microcapsules, implants, or wafers.

[0311] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable to high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. It is often preferable to include isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin. Furthermore, the compounds can be administered in time-release formulations, for example, compositions containing sustained-release polymers. The compounds can also be prepared with carriers that protect against rapid release, for example, controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid, polyglycol copolymers (PLG).

[0312] Many methods for preparing such formulations are generally known to those skilled in the art.Sterile injectable solution can be prepared by incorporating the active compound, such as the polypeptide or binding agent provided herein, in the required amount into a suitable solvent, which contains one or a combination of the above-listed ingredients, as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above.For sterile powders for preparing sterile injectable solution, the general preparation method is vacuum drying and freeze-drying, which obtains a powder of the active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.Compounds can also be formulated with one or more additional compounds that improve their solubility.

[0313] In some embodiments, pharmaceutical compositions of the present disclosure comprise sterile injectable solutions, hi some embodiments, sterile injectable solutions comprise sterile powders to be reconstituted with an acceptable solution (e.g., water).

[0314] It is often advantageous to formulate compositions (such as parenteral compositions) into unit dosage forms for ease of administration and uniformity of dosage. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other animals, each unit containing a predetermined amount of active substance calculated to produce a desired therapeutic effect, in association with a suitable pharmaceutical carrier. The specifications for the unit dosage forms of the present invention will vary, be influenced by, and be directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such therapeutic compounds for the prevention or treatment of TGFβ superfamily-related diseases or conditions. Dosages are discussed further below.

[0315] Dosage regimens will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient will depend on many factors, including the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly.

[0316] For any compound, the therapeutically effective dose can be initially estimated in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. Animal models can also be used to determine concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. These techniques are well known to those skilled in the art, and a therapeutically effective dose refers to the amount of active ingredient that ameliorates symptoms or conditions. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by calculating and controlling the ED50 (the dose therapeutically effective in 50% of the population) and the LD50 (the dose lethal to 50% of the population). Any of the pharmaceutical compositions described herein can be applied to any subject in need of treatment, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and especially humans.

[0317] The terms "effective dose," "effective dosage," and "effective amount" are used interchangeably to refer to an amount sufficient to achieve or at least partially achieve a desired effect. The term "therapeutically effective" dose or amount is defined as an amount sufficient to treat, ameliorate, or at least partially halt a disease and its complications in a patient already suffering from the disease. A therapeutically effective amount of a polypeptide or binding agent or pharmaceutical composition of the present disclosure generally results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease-free symptom-free periods, or prevention of functional impairment or disability resulting from disease affliction. In some embodiments, a therapeutically effective amount is an amount or dose of a polypeptide or binding agent or composition that prevents or treats a TGFβ superfamily-related disease or disorder in a subject, as described herein. In some embodiments, an effective amount is an amount or dose of a polypeptide or binding agent or composition that inhibits one or more of activin A, activin B, GDF-8, and GDF-11 in a subject, as described herein.

[0318] The amount or dosage effective for this use will depend on the disorder (indication) being treated, the polypeptide or binding agent being delivered, the treatment context and purpose, the severity of the disease, previous therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's size (weight, body surface, or organ size) and / or condition (age and general health), and the general state of the patient's own immune system. The appropriate dosage can be adjusted according to the judgment of the attending physician so that it can be administered to the patient once or over a series of administrations to obtain the optimal therapeutic effect.

[0319] Typical dosages may range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors mentioned above. In certain embodiments, dosages may range from 1.0 μg / kg to about 20 mg / kg, optionally 10 μg / kg to about 10 mg / kg, or 100 μg / kg to about 5 mg / kg. Dosages are discussed further below.

[0320] How to use The formulations described herein are useful as pharmaceutical compositions for treating, alleviating, and / or preventing diseases / disorders in subjects in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes application or administration of the formulations to the body, isolated tissue, or cells of a patient with a disease / disorder, symptoms of a disease / disorder, or predisposition toward a disease / disorder, with the purpose of curing, healing, alleviating, relieving, altering, repairing, ameliorating, improving, or affecting the disease, symptoms of a disease, or predisposition toward a disease.

[0321] References herein to methods of treatment may also be construed as references to the ActRIIB-ECD polypeptide described herein, or a TGFβ superfamily ligand binding agent comprising the same, for use in such methods of treatment.

[0322] The term "amelioration," as used herein, refers to any improvement in the disease state of a patient having a disease / disorder specified herein below, by administering to the subject a polypeptide or binding agent or pharmaceutical composition according to the present disclosure. For example, the term "amelioration" means reducing, suppressing, attenuating, arresting, or stabilizing the onset or progression of a disease. Such improvement may also be seen as a delay or halt in the progression of the patient's disease / disorder. The term "prevention," as used herein, means avoiding the occurrence or recurrence of a patient having a disease / disorder specified herein below, by administering to a subject in need thereof a polypeptide or binding agent or pharmaceutical composition according to the present disclosure.

[0323] The term "disease or condition" (or "disease / condition") refers to any pathological medical condition that may benefit from treatment with a polypeptide or binding agent or pharmaceutical composition described herein. This includes chronic and acute diseases or conditions, including pathological conditions that predispose a mammal to the disease / disorder in question. In some embodiments, the polypeptides or binding agents and compositions thereof of the present disclosure are useful for the prevention, treatment, or amelioration of a TGFβ superfamily-associated disease or condition. Accordingly, methods are provided for the prevention or treatment of a TGFβ superfamily-associated disease or condition in a subject, the methods comprising administering a therapeutically effective amount of a polypeptide or binding agent or pharmaceutical composition described herein. The polypeptides and binding agents are generally administered in the form of a pharmaceutical composition. The subject may be in need of such treatment, i.e., have, are suspected of having, or are at risk of having a disease or condition associated with TGFβ superfamily signaling or mediated by one or more members of the TGFβ superfamily, as described herein.

[0324] The term "inhibition" or "inhibiting" is used herein generally to refer to, but is not limited to, reducing, delaying, restricting, slowing, suppressing, blocking, neutralizing, impeding, or preventing a process, such as reducing or slowing the progression, growth, or spread of a disease or condition.

[0325] In some embodiments of the present disclosure, "treating" refers to neutralizing excess activin A, activin B, GDF-8, and / or GDF-11 biological activity, which may be determined by a suitable clinical parameter of improvement, a pathological assessment of the effect on the disease or condition, direct inhibition of activin A, activin B, GDF-8, and / or GDF-11 signaling (without substantially inhibiting BMP-9 and / or BMP-10), or another measure suitable for the disease or condition being treated.

[0326] In some embodiments, improvement is determined by comparing clinical variables to measurements taken before treatment, or alternatively, by comparing to typical values ​​measured in healthy adults. In some embodiments, treatment or prevention is within the context of the present disclosure if there is a measurable difference in the performance of a subject treated using the polypeptides, binding agents, compositions, and methods provided herein compared to members of a placebo group, compared to historical controls, or between subsequent tests administered to the same subject.

[0327] The term "subject" includes a living organism having, susceptible to, or at risk for a disease or condition associated with the TGFβ superfamily. Examples of subjects include mammals, such as humans, monkeys, cows, rabbits, sheep, goats, pigs, dogs, cats, rats, mice, and transgenic species thereof. The term "subject" generally includes animals, e.g., mammals, e.g., primates, e.g., humans, susceptible to a condition characterized by a TGFβ superfamily-associated disease or condition, such as pulmonary hypertension, fibrosis, muscle weakness and atrophy, metabolic and / or cardiometabolic disease, bone damage, or low red blood cell levels. The animal may also be an animal model of a disorder, e.g., a mouse model, a xenograft recipient, etc. In certain embodiments, the subject is a human.

[0328] There are no particular limitations on the dose of polypeptide or binding agent for use in the compositions and methods of the present disclosure. Exemplary doses include milligram or microgram amounts of polypeptide or binding agent per kilogram of subject or sample weight (e.g., about 50 micrograms to about 500 milligrams per kilogram, about 1 milligram to about 100 milligrams per kilogram, about 1 milligram to about 50 milligrams per kilogram, about 1 milligram to about 10 milligrams per kilogram, or about 3 milligrams to about 5 milligrams per kilogram). Additional exemplary doses include about 5 to about 500 mg, about 25 to about 300 mg, about 25 to about 200 mg, about 50 to about 150 mg, or about 50, about 100, about 150 mg, about 200 mg, or about 250 mg, and may be administered, for example, once or twice daily, or in lower or higher amounts.

[0329] In some embodiments, the dose range for an adult human is generally 0.005 mg to 10 g per day. The polypeptides or binding agents and compositions thereof may be provided in unit dosage form, e.g., in units effective for such dosages, or as multiples of such units, e.g., 5 mg to 500 mg, usually about 10 mg to 200 mg. Dosage units may be, for example, 1 to 30 mg, 1 to 40 mg, 1 to 100 mg, 1 to 300 mg, 1 to 500 mg, 2 to 500 mg, 3 to 100 mg, 5 to 20 mg, 5 to 100 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 The amount of the polypeptide or binding agent or composition described herein may be 25 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg).

[0330] It should be understood that the effective amount of polypeptide or binding agent for therapeutic treatment of a disease or condition varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. It should be understood that the dosage or amount of polypeptide or binding agent used alone or in combination with one or more administered active compounds will depend on each individual case and will routinely be adapted to individual circumstances to achieve optimal efficacy. Dosage and administration regimens are within the skill of those in the art, and the appropriate dose will usually depend on several factors within the knowledge of a physician, veterinarian, or researcher of ordinary skill in the art (see, for example, Wells et al. eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000)). For example, dosage and administration regimens will also depend on the nature and severity of the disease or condition being treated, as well as the sex, age, weight, and individual response of the human or animal being treated, the efficacy and duration of action of the compounds used, whether the treatment is acute, chronic, or prophylactic, and / or whether other active compounds are administered in addition to the therapeutic molecule(s).

[0331] Administration of the polypeptides or binding agents or compositions provided herein can be carried out using known procedures at dosages and for periods effective to achieve the desired purpose. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, weekly, biweekly, or monthly, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. In some embodiments, the polypeptides or binding agents or compositions are administered at a therapeutically effective dose sufficient to prevent or treat a TGFβ superfamily-related disease or condition in a subject, such as pulmonary hypertension, fibrosis, muscle weakness and atrophy, metabolic and / or cardiometabolic disorders, bone damage, and / or low red blood cell levels.

[0332] In some embodiments, according to the methods of the present disclosure, one or more symptoms of the onset or progression of a TGFβ superfamily-associated disease or condition are reduced in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0333] In some embodiments of the methods provided herein, the polypeptide or binding agent is administered in combination with one or more additional therapies or therapeutic agents. The additional therapies or therapeutic agents can be administered before, after, or simultaneously with administration of the polypeptide or binding agent or composition described herein. In some embodiments, the additional therapies or therapeutic agents are formulated with the polypeptide or binding agent in the same composition. In other embodiments, the additional therapies or therapeutic agents are administered separately. Examples of additional therapies and therapeutic agents include, but are not limited to, anti-fibrotic agents, anti-cancer agents, anti-inflammatory agents, anti-obesity agents, anti-diabetic agents, another TGFβ superfamily ligand binding agent or inhibitor, e.g., an antibody, antibody fragment, antigen-binding fragment, soluble TGFβ superfamily ligand trap, or another agent that binds to or inhibits one or more additional targets.

[0334] Alternatively, in some embodiments, the polypeptide or binding agent may be conjugated to a detectable or diagnostic moiety useful for tracking the polypeptide or binding agent or cells or tissues expressing a TGFβ superfamily ligand and / or another target. In some such embodiments, methods are provided for diagnosing a TGFβ superfamily-related disease or condition, comprising administering to a subject a polypeptide or binding agent of the present disclosure conjugated to a detectable or diagnostic moiety, and detecting the polypeptide or binding agent such that a disease or disorder associ...

Claims

1. A polypeptide comprising an amino acid sequence that is at least 85% identical to an activin receptor type IIB (ActRIIB) ectodomain (ECD) variant, said polypeptide comprising an amino acid substitution at a position corresponding to position 33 of SEQ ID NO:

2.

2. 10. The polypeptide of claim 1, wherein the ActRIIB ECD variant exhibits reduced inhibition of BMP-9 and / or BMP-10 compared to a wild-type ActRIIB ectodomain.

3. 3. The polypeptide of claim 1 or claim 2, wherein the amino acid substitution is selected from L33W, L33F, L33Q, L33Y, L33H, L33R, L33E, L33K, and L33M.

4. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33F.

5. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10; or (b) A polypeptide according to claim 4, comprising or consisting of the amino acid sequence of SEQ ID NO:

10.

6. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33Q.

7. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11; or (b) A polypeptide according to claim 6, comprising or consisting of the amino acid sequence of SEQ ID NO:

11.

8. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33Y.

9. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; or (b) A polypeptide according to claim 8, comprising or consisting of the amino acid sequence of SEQ ID NO:

12.

10. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33W.

11. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13; or (b) A polypeptide according to claim 10, comprising or consisting of the amino acid sequence of SEQ ID NO:

13.

12. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33H.

13. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14; or (b) A polypeptide according to claim 12, comprising or consisting of the amino acid sequence of SEQ ID NO:

14.

14. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33R.

15. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15; or (b) A polypeptide according to claim 14, comprising or consisting of the amino acid sequence of SEQ ID NO:

15.

16. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33E.

17. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16; or (b) A polypeptide according to claim 16, comprising or consisting of the amino acid sequence of SEQ ID NO:

16.

18. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33K.

19. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17; or (b) A polypeptide according to claim 18, comprising or consisting of the amino acid sequence of SEQ ID NO:

17.

20. The polypeptide of any one of claims 1 to 3, wherein the ActRIIB ECD variant comprises the amino acid substitution L33M.

21. the ActRIIB ECD variant is (a) comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18; or (b) a polypeptide according to claim 20, comprising or consisting of the amino acid sequence of SEQ ID NO:

18.

22. 22. The polypeptide of claim 21 , wherein the AtRIIB ECD variant further comprises an amino acid substitution at position 27 of SEQ ID NO:

2.

23. 23. The polypeptide of claim 21 or 22, wherein the AtRIIB ECD variant further comprises an amino acid substitution at position 69 of SEQ ID NO:

2.

24. 24. The polypeptide of any one of claims 22-23, wherein the ActRIIB-ECD variant further comprises one or more amino acid substitutions that are G27D, G27E, T69E, T69Q, or T69H.

25. 25. The polypeptide of any one of claims 1-24, wherein the ActRIIB ECD variant further comprises one or more additional amino acids at the N- or C-terminus.

26. 26. The polypeptide of claim 25, wherein the ActRIIB ECD variant further comprises the following amino acid at the N-terminus: GRGEA (SEQ ID NO: 63) and / or the following amino acid at the C-terminus: APT.

27. The polypeptide of any one of claims 1 to 26, further comprising an Fc domain monomer.

28. 28. The polypeptide of claim 27, further comprising a peptide linker positioned between the ActRIIB ECD variant and the Fc domain monomer.

29. 29. The polypeptide of claim 28, comprising, from N-terminus to C-terminus, the following structure: ActRIIB ECD-peptide linker-Fc domain monomer.

30. 30. The polypeptide of any one of claims 27 to 29, wherein the Fc domain monomer is of the IgG1, IgG2, IgG3 or IgG4 isotype.

31. The polypeptide of any one of claims 27 to 30, wherein the Fc domain monomer is a human Fc domain monomer or a mouse Fc domain monomer.

32. 32. The polypeptide of any one of claims 27 to 31, wherein the Fc domain monomers are engineered to reduce aggregation or modulate the stability of dimeric forms of the polypeptide.

33. 33. The polypeptide of claim 32, wherein the Fc domain monomer comprises the amino acid substitutions M252Y, S254T, and T256E (YTE).

34. 33. The polypeptide of claim 32, wherein the Fc domain monomer comprises the M252Y amino acid substitution.

35. 35. The polypeptide of any one of claims 30 to 34, wherein the Fc domain monomer comprises a D at position 356 and an L at position 358 (DL).

36. 35. The polypeptide of any one of claims 30 to 34, wherein the Fc domain monomer comprises E at position 356 and M(EM) at position 358.

37. The polypeptide of any one of claims 30 to 36, wherein the Fc domain monomer further comprises a lysine residue (K) at the C-terminus.

38. 38. The polypeptide of any one of claims 27-37, wherein the Fc domain monomer comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:252-292.

39. 39. The polypeptide of claim 38, wherein the Fc domain monomer comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 252-292.

40. 40. The polypeptide of any one of claims 30 to 39, wherein the Fc domain monomer is of the IgG1 isotype.

41. 41. The polypeptide of claim 40, wherein the Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO:253, SEQ ID NO:255, or SEQ ID NO:

256.

42. The polypeptide of any one of claims 27 to 41, wherein the Fc domain monomers form a dimer.

43. 43. The polypeptide of any one of claims 28 to 42, wherein the peptide linker is glycine-rich.

44. 44. The polypeptide of any one of claims 28 to 43, wherein the peptide linker is 10 to 40 amino acids in length.

45. 45. The polypeptide of claim 44, wherein the peptide linker is at least 10 amino acids in length, at least 14 amino acids in length, at least 19 amino acids in length, or at least 39 amino acids in length.

46. 46. ​​The polypeptide of claim 45, wherein the peptide linker is 10, 14, 19, or 39 amino acids in length.

47. 47. The polypeptide of claim 46, wherein the peptide linker is 14 amino acids in length.

48. 47. The polypeptide of claim 46, wherein the peptide linker is 19 amino acids in length.

49. 48. The polypeptide of any one of claims 43 to 47, wherein the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 89, 94, or 98.

50. 50. The polypeptide of any one of claims 1-49, wherein the ActRIIB-ECD comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 4-62.

51. 50. The polypeptide of any one of claims 1 to 49, wherein the ActRIIB-ECD comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 4 to 62.

52. 52. The polypeptide of any one of claims 1 to 51, wherein the ActRIIB-ECD comprises or consists of an amino acid sequence of SEQ ID NO: 10-18, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

53. 52. The polypeptide of any one of claims 1 to 51, wherein the ActRIIB-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 13, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to said amino acid sequence.

54. 54. The polypeptide of any one of claims 1 to 53, comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 174-251.

55. 55. The polypeptide of any one of claims 1 to 54, wherein the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 174 to 251.

56. 55. The polypeptide of any one of claims 1 to 54, comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234.

57. 55. The polypeptide of any one of claims 1 to 54, comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234.

58. 55. The polypeptide of any one of claims 1 to 54, comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 211 and 230-234.

59. 55. The polypeptide of any one of claims 1 to 54, wherein the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 211 and 230-234.

60. 55. The polypeptide of any one of claims 1 to 54, comprising or consisting of the amino acid sequence of SEQ ID NO: 231, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence.

61. 55. A polypeptide according to any one of claims 1 to 54, comprising or consisting of the amino acid sequence of SEQ ID NO:

231.

62. 55. The polypeptide of any one of claims 1 to 54, comprising or consisting of the amino acid sequence of SEQ ID NO: 234, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence.

63. 55. A polypeptide according to any one of claims 1 to 54, comprising or consisting of the amino acid sequence of SEQ ID NO:

234.

64. 64. The polypeptide of any one of claims 1 to 63, further comprising an albumin binding domain, a fibronectin domain, or a human serum albumin domain fused to the N-terminus or C-terminus of ActRIIB-ECD via a linker.

65. The polypeptide of any one of claims 1 to 64, further comprising a signal peptide of SEQ ID NO: 1 at the N-terminus of the ActRIIB-ECD.

66. 66. The polypeptide of claim 65, wherein the signal peptide is cleaved from the mature protein.

67. 67. The polypeptide of any one of claims 1 to 66, wherein the polypeptide is conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, or a diagnostic moiety.

68. 68. The polypeptide of claim 67, wherein the targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety comprises an antibody or antigen-binding fragment thereof, a binding agent having affinity for another member of the TGFβ superfamily or for another therapeutic target, a radiotherapeutic agent, an imaging agent, a fluorescent moiety, a cytotoxic agent, an antimitotic agent, a nanoparticle-based carrier, a polymeric binding agent, a nanocarrier, an imaging agent, a stabilizing agent, a drug, a nanocarrier, or a dendrimer.

69. 69. The polypeptide of any one of claims 1 to 68, which forms a dimer comprising a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide linked by at least one disulfide bond between an Fc domain monomer of the first polypeptide and an Fc domain monomer of the second polypeptide.

70. 70. A TGFβ superfamily ligand binding agent comprising a first polypeptide according to any one of claims 1 to 69 and a second polypeptide according to any one of claims 1 to 69, wherein the first polypeptide and the second polypeptide are linked by at least one disulfide bond between the Fc domain monomer of the first polypeptide and the Fc domain monomer of the second polypeptide.

71. 71. The binding agent of claim 70, wherein the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 174-251, or an amino acid sequence which is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to said amino acid sequence.

72. 71. The binding agent of claim 70, wherein the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214, and 230-234, or an amino acid sequence which is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to said amino acid sequence.

73. 71. The binding agent of claim 70, wherein the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 211 and 230-234, or an amino acid sequence which is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to said amino acid sequence.

74. 71. The binding agent of claim 70, wherein the first polypeptide and the second polypeptide comprise or consist of SEQ ID NO:231, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

231.

75. 71. The binding agent of claim 70, wherein the first polypeptide and the second polypeptide comprise or consist of SEQ ID NO:234, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

234.

76. 71. The binding agent of claim 70, wherein the first polypeptide and the second polypeptide comprise or consist of SEQ ID NO:211, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

211.

77. A polypeptide of any one of claims 1 to 69 or a binding agent of any one of claims 70 to 75, wherein the polypeptide and / or binding agent exhibits similar or increased binding to human activin A, activin B, GDF-8, and / or GDF-11, and decreased binding to human BMP-9 and / or BMP-10, compared to a polypeptide comprising wild-type ActRIIB-ECD.

78. 76. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 75, which does not substantially bind to human BMP-9.

79. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, which exhibits reduced binding to human BMP-10 compared to a polypeptide comprising wild-type ActRIIB-ECD.

80. A polypeptide according to any one of claims 1 to 69 or a binding agent according to any one of claims 70 to 75, which inhibits signaling of one or more of human activin A, activin B, GDF-8, and GDF-11.

81. 77. A polypeptide according to any one of claims 1 to 69 or a binding agent according to any one of claims 70 to 76, which does not substantially inhibit human BMP-9 and / or BMP-10 signaling.

82. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against human BMP-9 and / or BMP-10 signaling is reduced by about 5-fold, about 10-fold, or about 100-fold or more compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 and / or BMP-10 signaling.

83. A polypeptide described in any one of claims 1 to 69 or a binding agent described in any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against human BMP-9 and / or BMP-10 signaling is reduced by about 200-fold, about 300-fold or more compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 and / or BMP-10 signaling.

84. A polypeptide described in any one of claims 1 to 69 or a binding agent to any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against human BMP-9 and / or BMP-10 signaling is reduced by about 5-fold, about 10-fold, or about 100-fold or more compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against human BMP-9 and / or BMP-10 signaling.

85. A polypeptide described in any one of claims 1 to 69 or a binding agent described in any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against human BMP-9 and / or BMP-10 signaling is reduced by about 200-fold, about 300-fold or more compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO:

171.

86. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against one or more of human activin A, activin B, GDF-8, and GDF-11 is the same or substantially the same as the inhibitory potency of human wild-type ActRIIB-ECD against the same respective ligand(s).

87. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the inhibitory potency of said polypeptide against one or more of human activin A, activin B, GDF-8, and GDF-11 is the same or substantially the same as the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 57 against the same respective ligand(s).

88. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the relative inhibitory potency against one or more of human activin A, activin B, GDF-8, and GDF-11 is increased compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same respective ligand(s), and / or the relative inhibitory potency against BMP-9 and / or BMP-10 is reduced compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same ligand(s).

89. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against one or more of human activin A, activin B, GDF-8, and GDF-11 is increased compared to the inhibitory potency against the respective ligand(s), and / or the relative inhibitory potency against BMP-9 and / or BMP-10 is decreased compared to the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same ligand.

90. 77. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the relative inhibitory potency against one or more of human activin A, activin B, GDF-8, and GDF-11 is increased by about 2-fold or more, about 3-fold or more, about 4-fold or more, or about 5-fold or more compared to the inhibitory potency of human wild-type ActRIIB-ECD or the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same respective ligand(s).

91. A polypeptide described in any one of claims 1 to 69 or a binding agent described in any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against activin A is at least about 2-fold higher than the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 57, and the inhibitory potency of the polypeptide against BMP-9 and / or BMP-10 is at least about 10-fold lower than the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO:

171.

92. (a) the inhibitory potency of the polypeptide against activin A is at least about 5-fold higher than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the relative inhibitory potency against BMP-9 and / or BMP-10 is at least about 100-fold lower than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171; (b) the inhibitory potency of the polypeptide against activin B is at least about 5-fold greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the relative inhibitory potency against BMP-9 and / or BMP-10 is at least about 100-fold less than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171; or (c) a polypeptide described in any one of claims 1 to 69 or a binding agent described in any one of claims 70 to 76, wherein the inhibitory potency of the polypeptide against both activin A and activin B is at least about 5 times higher than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171, and the relative inhibitory potency against BMP-9 and / or BMP-10 is at least about 100 times lower than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO:

171.

93. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 69.

94. 94. The nucleic acid molecule of claim 93, further comprising the sequence set forth in SEQ ID NO: 297 at the 5' end of the nucleic acid molecule.

95. A vector comprising the nucleic acid of claim 93 or 94.

96. 96. A host cell comprising a nucleic acid molecule of any one of claims 93 or 94 or a vector of claim 95, wherein the nucleic acid molecule or vector is expressed internally.

97. A method for preparing a polypeptide according to any one of claims 1 to 69, comprising the steps of: (a) providing a host cell comprising the nucleic acid molecule of any one of claims 93 or 94 or the vector of claim 95; (b) culturing the host cell under conditions that allow expression of the polypeptide; (c) recovering the expressed polypeptide from the culture.

98. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 69 or a binding agent according to any one of claims 70 to 92, and a pharmaceutically acceptable carrier, diluent or excipient.

99. 99. The pharmaceutical composition of claim 98, formulated for administration by injection or infusion.

100. 100. The pharmaceutical composition of claim 99, formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

101. 101. The pharmaceutical composition of any one of claims 98 to 100, wherein the polypeptide or binding agent does not cause vascular complications in a subject and / or does not increase vascular permeability or leakage in a subject.

102. 102. The pharmaceutical composition of any one of claims 98-101, wherein the polypeptide or binding agent does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and / or does not cause hematological complications in a subject.

103. 102. A kit comprising a polypeptide according to any one of claims 1 to 69, a binding agent according to any one of claims 70 to 92, or a pharmaceutical composition according to any one of claims 98 to 102, and optionally instructions for use.

104. 102. A method for treating or preventing a disease or condition associated with signaling of a TGFβ superfamily ligand in a subject in need thereof, the method comprising administering to the subject a polypeptide according to any one of claims 1 to 69, a binding agent according to any one of claims 70 to 92, or a pharmaceutical composition according to any one of claims 98 to 102.

105. 105. The method of claim 104, wherein the subject is a human.

106. 106. The method of claim 104 or 105, wherein the TGFβ superfamily ligand is one or more of activin A, activin B, GDF-8, and GDF-11.

107. A method of treating or preventing a disease or condition mediated by activin A, activin B, GDF-8, and / or GDF-11 in a subject, the method comprising administering to the subject a polypeptide described in any one of claims 1 to 69, a binding agent described in any one of claims 70 to 92, or a pharmaceutical composition described in any one of claims 98 to 102.

108. 108. The method of claim 107, wherein the disease or condition is characterized by overexpression or overactivation of activin A and / or activin B and / or GDF-8 and / or GDF-11.

109. 109. The method of any one of claims 104 to 108, wherein the disease or condition is selected from pulmonary hypertension (PH), fibrosis, muscle weakness or atrophy, metabolic disorders, cardiometabolic diseases, bone damage, and low red blood cell levels.

110. 110. The method of claim 109, wherein the PH is pulmonary arterial hypertension (PAH).

111. 111. The method of claim 110, wherein the PAH is idiopathic PAH, hereditary PAH, or PAH associated with an infection, congenital heart abnormality, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorder, chronic obstructive pulmonary disease, autoimmune disorder (e.g., scleroderma or lupus), or drug use (e.g., cocaine or methamphetamine use).

112. 110. The method of claim 109, wherein the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, kidney fibrosis, myelofibrosis, systemic sclerosis, dermal fibrosis, cardiac fibrosis, myelofibrosis, corneal fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthrofibrosis, arthrofibrosis, tissue fibrosis, a fibromatous proliferative disorder, or a connective tissue disorder.

113. 110. The method of claim 109, wherein the muscle weakness or wasting disease or condition is Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia.

114. 110. The method of claim 109, wherein the metabolic disorder is obesity, type 1 diabetes, type 2 diabetes, or prediabetes.

115. 115. The method of claim 114, wherein the metabolic disorder is obesity.

116. 110. The method of claim 109, wherein the cardiometabolic disease or condition is heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

117. The method of claim 109, wherein the bone damage comprises bone demineralization, osteoporosis (e.g., primary or secondary), osteopenia, osteopetrosis, fracture, bone loss associated with bone cancer or cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobility-related bone loss.

118. 110. The method of claim 109, wherein the disease or condition of low blood cell levels is anemia or blood loss.

119. 102. A method for reducing or inhibiting activin A, activin B, GDF-8, and / or GDF-11 signaling in a subject in need thereof, without substantially reducing or inhibiting BMP-9 and / or BMP-10 signaling in the subject, the method comprising administering to the subject a polypeptide described in any one of claims 1 to 69, a binding agent described in any one of claims 70 to 92, or a pharmaceutical composition described in any one of claims 98 to 102.

120. 120. The method of claim 119, wherein the subject is a mammal.

121. 121. The method of claim 120, wherein the mammal is a human.

122. 122. The method of any one of claims 104 to 121, wherein the method does not cause vascular complications in a subject, does not increase vascular permeability or leakage in a subject, does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and / or does not cause hematologic complications in a subject.

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