Activin IIB type receptor variants and uses thereof

Abstract

The present disclosure provides polypeptides comprising an activin type IIB receptor (ActRIIB) extracellular domain (ECD) variant. In some embodiments, the polypeptides of the present disclosure include an ActRIIB-ECD variant fused to an Fc domain moiety. The disclosure also provides pharmaceutical compositions and methods of using the polypeptides in the treatment of diseases and conditions associated with TGF [beta] superfamily ligand signaling, such as metabolic disorders, diabetes, obesity, cardiac metabolic disease, pulmonary hypertension, fibrosis, muscle weakness and atrophy, bone injury, and / or low red blood cell levels, such as anemia.

Description

[0001] Cross Reference to Related Applications

[0002] This is an international PCT application claiming priority to U.S. Provisional Application No. 63 / 434,576, filed December 22, 2022; U.S. Provisional Application No. 63 / 443,015, filed February 2, 2023; U.S. Provisional Application No. 63 / 500,881, filed May 8, 2023; U.S. Provisional Application No. 63 / 507,638, filed June 12, 2023; and U.S. Provisional Application No. 63 / 515,562, filed July 25, 2023, the contents of each of which are incorporated herein by reference.

[0003] Incorporation by Reference of Electronic Sequence Listing

[0004] The content of the electronic sequence listing (35PH_003_05WO_SeqList_ST26.xml; size: 440,400 bytes; and creation date: December 18, 2023) is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0005] The present disclosure relates to polypeptides comprising Activin Receptor Type IIB (ActRIIB) extracellular domain (ECD) variants and uses of the polypeptides for binding and neutralizing TGFβ superfamily ligands, particularly for treating diseases and conditions associated with TGFβ superfamily signaling, such as metabolic disorders, cardiometabolic diseases, pulmonary hypertension, fibrosis, muscle weakness and atrophy, bone injury, and low red blood cell levels. BACKGROUND

[0006] The transforming growth factor β (TGFβ) superfamily comprises 35 ligands that regulate a variety of physiological processes, including cell proliferation, migration, and differentiation, muscle growth, vascular homeostasis, and osteogenesis. Perturbations in the levels and / or signaling pathways of the TGFβ superfamily can cause severe pathological effects. For example, TGFβ and Activin ligands are associated with the pathogenesis of a variety of human disorders and play a key pathogenic role in many diseases. Examples of TGFβ superfamily-associated disorders include metabolic and cardiometabolic disorders (including diabetes and obesity), pulmonary hypertension (including pulmonary arterial hypertension), hematological malignancies, solid tumors, bone marrow failure states, muscle weakness, and a variety of disorders characterized by uncontrolled fibrosis, such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, 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 therapeutics that effectively treat TGFβ superfamily-associated disorders. SUMMARY

[0007] Provided herein are activin type IIB receptor (ActRIIB) extracellular domain (ECD)-based traps and pharmaceutical compositions with tailored ligand-specificity characteristics for binding and neutralizing TGFβ superfamily ligands, and methods of their use in treating diseases and conditions associated with or mediated by TGFβ superfamily signaling.

[0008] The ActRIIB-ECD capture agent provided herein comprises an ActRIIB-ECD variant fused to an Fc domain monomer, which can act to assemble two polypeptides together. The ActRIIB-ECD variant provided herein is designed to customize ligand specificity in order to maximize therapeutic efficacy in certain disease indications while minimizing adverse reactions. The ActRIIB-ECD variant provided herein is constructed by introducing novel amino acid substitutions into ActRIIB-ECD with the aim of preventing or reducing the interruption 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 being limited by theory, the goal of retaining 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; therefore, ActRIIB-ECD-based traps have the potential to disrupt vascular homeostasis, leading to bleeding problems. In support of this concept, telangiectasia, epistaxis, and gingival bleeding were observed in clinical studies of a non-mutant ActRIIB-ECD trap (called ACE-031) (Campbell, C. et al., 2017). This suggests that these vascular effects may be caused by inhibition of the BMP-9 pathway.

[0009] 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, which enables them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous ligand-stimulated receptor signaling; and (2) reduced or abolished binding to BMP-9 and / or BMP-10 compared to wild-type ActRIIB, which enables maintenance of steady-state 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 miscellaneous PH), metabolic diseases, bone diseases, muscle diseases, fibrosis, and / or low red blood cell levels (e.g., anemia). The variants can, for example, result in a reduction in symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or mixed 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 adipose tissue, a reduction in fibrosis (e.g., a reduction in fibrosis or a slowing or halting of fibrosis progression), and / or an increase in red blood cell levels (e.g., an increase in hemoglobin level, hematocrit, or red blood cell count).

[0010] In some embodiments, the present disclosure provides a polypeptide comprising an amino acid sequence at least 85% identical to an activin type IIB receptor extracellular domain (ECD) variant, the ActRIIB ECD variant 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 the wild-type ActRIIB extracellular domain. In some embodiments, the amino acid substitution is selected from L33F, L33Q, L33Y, L33W, L33H, L33R, L33E, L33K, and L33M.

[0011] In some embodiments, the present disclosure provides a polypeptide comprising an amino acid sequence at least 90% identical to an activin type IIB receptor (ActRIIB) extracellular domain (ECD) variant, the ActRIIB ECD variant 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 the wild-type ActRIIB extracellular domain. In some embodiments, the amino acid substitution is selected from L33F, L33Q, L33Y, L33W, L33H, L33R, L33E, L33K, and L33M.

[0012] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33F. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33Q. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0014] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33Y. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0015] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33W. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0016] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33H. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0017] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33R. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0018] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33E. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0019] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33K. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0020] In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution L33M. In some embodiments, the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0021] In some embodiments, the ActRIIB ECD variant further comprises an amino acid substitution at position 27 of SEQ ID NO: 2. In some embodiments, the ActRIIB ECD variant further comprises an amino acid substitution at position 69 of SEQ ID NO: 2. In some embodiments, the ActRIIB ECD variant further comprises one or more amino acid substitutions, namely, G27D, G27E, T69E, T69Q, or T69H.

[0022] In some embodiments, the ActRIIB ECD variant further comprises one or more additional amino acids at the N-terminus or C-terminus. In some embodiments, the ActRIIB ECD variant further comprises the following amino acids at the N-terminus: GRGEA (SEQ ID NO: 63) and / or the following amino acids at the C-terminus: APT.

[0023] 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.

[0024] In some embodiments, the Fc domain monomer is of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc domain monomer is a human Fc domain monomer or a murine Fc domain monomer.

[0025] In some embodiments, the Fc domain monomer is engineered to reduce the aggregation of dimers of the polypeptide or to regulate the stability of the dimer of the polypeptide. In some embodiments, the Fc domain monomer comprises amino acid substitutions M252Y, S254T, and T256E (YTE). In some embodiments, the Fc domain monomer comprises an M252Y amino acid substitution. 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.

[0026] 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 an IgG1 isotype. In some embodiments, the Fc domain monomer comprises or consists of an amino acid sequence selected from SEQ ID NOs: 253, 255, or 256.

[0027] In some embodiments, Fc domain monomers form dimers.

[0028] In some embodiments, the peptide linker is rich in glycine. In some embodiments, the length of the peptide linker is between 10 and 40 amino acids. In some embodiments, the length of the linker is at least 10 amino acids, at least 14 amino acids, at least 19 amino acids, or at least 39 amino acids in length. In some embodiments, the length of the linker is 10 amino acids, at least 14 amino acids, at least 19 amino acids, or at least 39 amino acids in length. In some embodiments, the length of the linker is 14 amino acids. In some embodiments, the length of the linker is 19 amino acids. In some embodiments, the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NO: 89, 94 or 98.

[0029] In some embodiments, the ActRIIB-ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 4-62. In some embodiments, the ActRIIB-ECD comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 4-62. In some embodiments, the 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 thereto. In some embodiments, the ActRIIB-ECD comprises or consists of an amino acid sequence of SEQ ID NOs: 13, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. 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: 174-251. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 174-251.

[0030] 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.

[0031] 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: 211 and 230-234. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 211 and 230-234. In some embodiments, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 231, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 231. In some embodiments, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 234, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 234.

[0032] 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 the 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 the ActRIIB-ECD. In some embodiments, the signal peptide is cleaved from the mature protein.

[0033] In some embodiments, the polypeptide is conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, or a diagnostic moiety. In some embodiments, the targeting agent, the therapeutic moiety, the detectable moiety, or the 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 drug, a nanoparticle-based carrier, a polymer-conjugated drug, a nanocarrier, an imaging agent, a stabilizer, a drug, a nanocarrier, or a dendrimer.

[0034] In some embodiments, the polypeptide forms a dimer comprising a first and a second polypeptide, the first polypeptide and the second polypeptide being 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.

[0035] 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.

[0036] 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 at least 95%, 96%, 97%, 98% or 99% identical thereto.

[0037] 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 at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0038] In some embodiments, 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 at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first polypeptide and the second polypeptide comprise or consist of SEQ ID NO: 231, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first polypeptide and the second polypeptide comprise or consist of SEQ ID NO: 234, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first polypeptide and the second polypeptide comprise or consist of SEQ ID NO: 211, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0039] 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 exhibits reduced binding to human BMP-9 and / or BMP-10, compared to a polypeptide comprising WT ActRIIB-ECD. In some embodiments, the polypeptide and / or binding agent does not substantially bind to human BMP-9.

[0040] In some embodiments, the polypeptide and / or binding agent exhibits reduced binding to human BMP-10 compared to a polypeptide comprising WT 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 and / or binding agent does not substantially inhibit human BMP-9 and / or BMP-10 signaling.

[0041] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency on human BMP-9 and / or BMP-10 signaling 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 on human BMP-9 and / or BMP-10 signaling.

[0042] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency on human BMP-9 and / or BMP-10 signaling that is reduced by about 200-fold, about 300-fold, or more compared to the inhibitory potency of human wild-type ActRIIB-ECD on human BMP-9 and / or BMP-10 signaling.

[0043] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency on human BMP-9 and / or BMP-10 signaling that 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.

[0044] In some embodiments, the polypeptide and / or binding agent has an inhibitory potency of about 200-fold, about 300-fold, or more reduced against human BMP-9 and / or BMP-10 signaling compared to the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.

[0045] In some embodiments, the polypeptide and / or binding agent inhibits one or more of human activin A, activin B, GDF-8, and GDF-11 with the same or substantially the same potency as human wild-type ActRIIB-ECD inhibits the same corresponding ligand.

[0046] In some embodiments, the polypeptide and / or binding agent inhibits one or more of human activin A, activin B, GDF-8, and GDF-11 with the same or substantially the same potency as the polypeptide having the amino acid sequence set forth in SEQ ID NO: 57 inhibits the same corresponding ligand.

[0047] In some embodiments, the relative inhibitory potency of the polypeptide and / or binding agent 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 corresponding ligands; and / or wherein the relative inhibitory potency against BMP-9 and / or BMP-10 is decreased compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same ligands.

[0048] In some embodiments, the relative inhibitory potency of the polypeptide and / or binding agent against one or more of human activin A, activin B, GDF-8, and GDF-11 is increased compared to the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same corresponding ligands; and / or wherein 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 ligands.

[0049] In some embodiments, the inhibitory potency of the polypeptide and / or binding agent 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 relative 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 corresponding ligand.

[0050] In some embodiments, the relative inhibitory potency of the polypeptide and / or binding agent against activin A is at least about 2-fold greater 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 less than the inhibitory potency of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171.

[0051] In some embodiments, the polypeptide and / or binding agent comprises: (a) an inhibitory potency of at least about 5 times greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for activin A, and an inhibitory potency of the polypeptide to BMP-9 and / or BMP-10 that is at least about 100 times lower than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for B; (b) an inhibitory potency of at least about 5 times greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for activin B, and an inhibitory potency of the polypeptide to BMP-9 and / or BMP-10 that is at least about 100 times lower than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for B; or (c) an inhibitory potency of at least about 5 times greater than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 for activin A and activin B, and an inhibitory potency of the polypeptide to BMP-9 and / or BMP-10 that is at least about 100 times lower than that of a polypeptide having the amino acid sequence set forth in SEQ ID NO: The inhibitory potency of the polypeptide having the amino acid sequence set forth in 171 was at least about 100-fold lower.

[0052] In some embodiments, the present disclosure provides a nucleic acid molecule encoding a polypeptide as 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.

[0053] In some embodiments, the present disclosure provides a vector comprising a nucleic acid molecule described herein.

[0054] In some embodiments, the present disclosure provides a host cell comprising a nucleic acid molecule or a vector described herein, wherein the nucleic acid molecule or the vector is expressed in the host cell.

[0055] In some embodiments, the present disclosure provides a method of preparing a polypeptide described herein, the method comprising: providing a host cell comprising a nucleic acid molecule or vector described herein; and culturing the host cell under conditions that allow expression of the polypeptide; and recovering the expressed polypeptide from the culture.

[0056] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a polypeptide or binding agent as 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.

[0057] In some embodiments, the polypeptide or binding agent does not cause vascular complications in the subject and / or does not increase vascular permeability or leakage in the subject. In 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 hematological complications in the subject.

[0058] In some embodiments, the present disclosure provides a kit comprising a polypeptide, binding agent, or pharmaceutical composition described herein, and optionally instructions for use.

[0059] In some embodiments, the present disclosure provides a method for 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 as described herein. In some embodiments, the subject is human. In some embodiments, the TGFβ superfamily ligand is one or more of activin A, activin B, GDF-8, and GDF-11.

[0060] In some embodiments, the present disclosure provides a method for 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 as 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 diseases, bone damage, and low red blood cell levels.

[0061] 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 anomaly, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, a connective tissue disorder, chronic obstructive pulmonary disease, an autoimmune disorder (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, liver fibrosis, lung fibrosis, kidney fibrosis, bone marrow fibrosis, systemic sclerosis, skin fibrosis, cardiac fibrosis, myelofibrosis, corneal fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthritis, arthrofibrosis, tissue fibrosis, a fibroproliferative disorder, or a connective tissue disorder. In some embodiments, 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. 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, bone damage includes bone demineralization, osteoporosis (e.g., primary or secondary), osteopenia, osteosclerosis, fracture, bone cancer or metastasis-related bone loss, 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. In some embodiments, the low blood cell level disease or disorder is anemia or blood loss.

[0062] 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 as 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 hematological complications in the subject.

[0063] Further scope, applicability, and advantages of the present technology will become apparent from the non-limiting detailed description given hereinafter. It should be understood, however, that the detailed description, while indicating exemplary embodiments of the present technology, is given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0065] To better understand the present technology and to more clearly demonstrate how it may be practiced, reference will now be made by way of example to the accompanying drawings which illustrate aspects and features according to non-limiting embodiments of the present technology.

[0066] Figures 1A-1B Polyacrylamide gel electrophoresis analysis of representative ActRIIB-ECD polypeptide constructs under non-reducing and reducing conditions is shown. 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; P1409: protein 1409. “NR”: non-reducing conditions; “R”: reducing conditions.

[0067] Figure 2A – Figure 2QA comparative chart (radar chart) is shown showing the IC50 values ​​of exemplary test proteins for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10), as indicated. Points at the center of the chart indicate low neutralization potency (high IC50 value) for a given cytokine, while points at the edge of the chart indicate high neutralization potency (low IC50 value) for a given cytokine. The exemplary test proteins shown are ( Figure 2A ) P739, P750 and P751; ( Figure 2B )P753, P754 and P1229;( Figure 2C ) P1373, P1371 and P1375; ( Figure 2D ) P1182 and P1185; and ( Figure 2E ) P1389, P1406, and P1409. All agents were compared to wild-type ActRIIB-ECD (P75). Figure 2F-2I Representative results are shown for exemplary proteins P75, P1229, P1371, P1372, P1373, P1374, and P1375 inhibiting activin A, activin B, GDF-8, and GDF-11, respectively, in a HEK-Blue cell-based assay. Figure 2J-2K Shown are representative results for the inhibition of BMP-9 and BMP-10, respectively, by exemplary proteins P75, P1229, P1372, P1373, P1374, and P1375 in a HepG2 cell-based assay. Figure 2L-Figure 2O Representative results are shown for the inhibition of activin A, activin B, GDF-8, and GDF-11 by exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486, respectively, in a HEK-Blue cell-based assay. Figure 2P-2Q Representative results are shown for the inhibition of BMP-9 and BMP-10 by exemplary proteins P75, P1229, P1373, P1483, P1484, P1485, and P1486, respectively, in a HepG2 cell-based assay. Error bars represent standard error of the mean (SEM).

[0068] Figure 3 Shown are the results of an activin A ELISA performed using the supernatant of a small-scale production ActRIIB-ECD fusion protein. Results are expressed as a % of the signal obtained using wild-type ActRIIB-ECD-Fc (P75). In this assay, loss of signal indicates increased binding of the exemplary agent to activin A. Error bars represent standard error of the mean (SEM). This figure was generated using GraphPad Prism 9.0.

[0069] Figure 4 Results from a BMP-9 ELISA using supernatant from small-scale production of ActRIIB-ECD fusion proteins are shown. Results are expressed as % of the signal obtained using wild-type ActRIIB-ECD-Fc (P75). In this assay, a loss of signal indicates that the exemplary test agent binds less to BMP-9. Error bars represent the standard error of the mean (SEM). These graphs were generated using GraphPad Prism 9.0.

[0070] Figure 5A Figure 5B Results from a BMP-10 assay using supernatant from small-scale production of ActRIIB-ECD fusion proteins are shown. Figure 5A Results from a cell-based assay are expressed as % of the signal obtained using BMP-10 alone. In this assay, a loss of signal indicates that the exemplary test agent binds more to BMP-10. Error bars represent the standard error of the mean (SEM). Figure 5B KD values for exemplary test agents were derived using bio-layer interferometry. These graphs were generated using GraphPad Prism 9.0.

[0071] Figure 6 Results from a wild-type mouse single injection experiment are shown. Male mice were injected with a single dose of test agent (25 mg / kg, subcutaneously) as indicated and body weight gain was assessed 4 days later. Results were normalized to the vehicle control and error bars represent the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni correction for multiple comparisons; relative to the vehicle group, p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

[0072] Figure 7A Figure 7B Body weight gain in male mice injected (subcutaneously) with test proteins is shown. Figure 7A Results for P750 (1, 5, or 25 mg / kg) are shown, and Figure 7B Results for P1229 (5 or 25 mg / kg) and P75 (25 mg / kg) are shown. Injections were given twice per week for 11 days (P750) or 7 days (P1229 and P75). Results were normalized to the vehicle control and error bars represent the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni correction for multiple comparisons; relative to the vehicle group, p < 0.05, ​​p<0.001, p<0.0001.

[0073] Figure 8 Exemplary schematic diagrams of the binding agents described herein are provided.

[0074] Figure 9A – Figure 9D Shown are changes in skeletal muscle mass after one week of treatment with vehicle, P1229 (5 or 25 mg / kg), and P75 (25 mg / kg). Subcutaneous injections were given twice weekly. Figure 9A Mean tibialis anterior muscle weights are shown. Figure 9B Tibialis muscle weights normalized to vehicle control group are shown. Figure 9C Mean gastrocnemius muscle weights are shown. Figure 9D Gastrocnemius muscle weights normalized to the vehicle control group are shown. Results were analyzed by one-way ANOVA followed by a post hoc Bonferroni correction for multiple comparisons; p<0.05, p<0.001.

[0075] Figure 10 The results show that the expression of P1229 in the tibialis anterior muscle increased after one week of treatment with vehicle, P1229 (5 or 25 mg / kg) and P75 (25 mg / kg). Mss51 The results were analyzed by one-way ANOVA followed by Bonferroni correction for multiple comparisons; compared with the vehicle group, p<0.0001.

[0076] Figure 11A – Figure 11F Depicted are changes in body composition in diet-induced obese (DIO) mice injected subcutaneously 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 bimalumab (an antibody targeting ActRIIB and ActRIIA), 20 mg / kg once weekly). Figure 11A and 11D Shows average lean body mass, Figure 11B and 11E The average fat mass is shown, and Figure 11C and 11F The ratio of lean body mass to fat body mass is shown. Parameters were assessed by echoMRI. Results were analyzed by one-way ANOVA followed by post hoc Bonferroni correction for multiple comparisons; p <0.05.

[0077] Figure 12A – Figure 12B Shown is food intake over time in DIO mice injected subcutaneously 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 bimalumab, 20 mg / kg once weekly). Figure 12C -H shows the skeletal muscle weight at the end of the study; Figure 12C – Figure 12D 、 Figure 12E – Figure 12F and Figure 12G – Figure 12H Tibialis anterior, gastrocnemius, and soleus muscle weights are shown separately. Results were analyzed by one-way ANOVA followed by post-hoc Bonferroni correction for multiple comparisons; p <0.05, p <0.01.

[0078] Figure 13A – Figure 13C Depicted are 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 bimalumab, 20 mg / kg once weekly). Figure 13A – Figure 13B The gastrocnemius muscle Mss51 Express. Figure 13C Explains the soleus muscle Serpine1 and Id1 The results were analyzed by one-way ANOVA followed by Bonferroni correction for multiple comparisons; relative to the vehicle group, p <0.05, p <0.01, p <0.001.

[0079] Figure 14A – Figure 14FDepicted are changes in liver gene expression in DIO mice following 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 bimalumab, 20 mg / kg once weekly). Figure 14A – Figure 14B Fetuin A is shown Ahsg Gene expression. Figure 14C – Figure 14D The gene encoding FGF21 is shown Fgf21 Gene expression. Figure 14E – Figure 14F The gene encoding activin E is shown Inhbe Gene expression. Results were analyzed by one-way ANOVA followed by Bonferroni correction for multiple comparisons; p < 0.05 relative to the vehicle group, p <0.01, p <0.001.

[0080] Figure 15A – Figure 15B Figure 2: Changes in follicle-stimulating hormone (FSH) production in DIO mice following 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 bimalumab, 20 mg / kg once weekly). Results were analyzed by one-way ANOVA followed by a post hoc Bonferroni correction for multiple comparisons; differences were statistically significant relative to vehicle. p <0.001, p <0.0001. DETAILED DESCRIPTION

[0081] Overview

[0082] Activin type II receptor is a single transmembrane domain receptor that regulates the signal of TGFβ superfamily ligands. 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) (e.g., BMP-9, BMP-10). The activity of TGFβ superfamily ligands is associated with a variety of diseases and conditions, including pulmonary hypertension (PH), fibrosis, muscle disorders (including muscular dystrophy), metabolic disorders (including type II diabetes), bone disease and anemia.

[0083] One approach to developing therapeutic agents that inhibit the function of TGFβ superfamily ligands is to use soluble decoy receptors (also known as ligand traps based on the extracellular domain (ECD) of the receptor) to bind and sequester the ligand, thereby preventing access to the cell surface receptor. In general, receptor ECD-based traps are a class of therapeutic agents that can selectively sequester ligands and can be optimized using protein engineering methods. For example, polypeptide fusions based on the extracellular domain of the TGFβ receptor that bind or "trap" TGFβ1 and / or TGFβ2 and / or TGFβ3 ligand isoforms have been used to inhibit TGFβ signaling (see, e.g., WO01 / 83525; WO2005 / 028517; WO2008 / 113185; WO2008 / 157367; WO2010 / 0031168; WO2010 / 099219; WO2012 / 0 71649; WO2012 / 142515; WO2013 / 000234; WO2018 / 158727; US5693607; US2005 / 0203022; US2007 / 0244042; US8318135; US8658135; US8815247; US2015 / 0225483; US2015 / 0056199; and WO2017 / 037634).

[0084] In the lung endothelium and blood vessels, bone morphogenetic proteins (BMPs) induce antiproliferative effects on smooth muscle cells (SMCs) and survival of endothelial cells (ECs), while activins and growth differentiation factors (GDFs) induce opposing effects, namely, proproliferative effects on SMCs and apoptosis in ECs (Yung, LM et al., 2020; Ryanto, GRT et al., 2021). Under physiological conditions, these ligands act synergistically to maintain homeostasis. However, in certain disease conditions, such as PAH, these pathways become unbalanced. For example, nearly 80% of familial PAH cases and approximately 20% of idiopathic PAH cases 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 would be desirable to provide a trap based on the extracellular domain of the receptor that could neutralize some ligands but not others, thereby rebalancing the pathway and re-establishing vascular homeostasis.

[0085] Thus, the present application provides TGFβ superfamily ligand binding agents that exhibit improved ligand binding characteristics and therapeutic efficacy.

[0086] The present technology is explained in more detail below. This description is not intended to be an exhaustive list of all different ways in which the present technology can be implemented, or an exhaustive list of all features that can be added to the present technology. For example, features described for one embodiment can be incorporated into other embodiments, and features described for a specific embodiment can be deleted from that embodiment. In addition, in view of this disclosure, it will be clear to those skilled in the art that many variations and additions of the various embodiments proposed herein do not depart from the present technology. Therefore, the following description is intended to illustrate some specific embodiments of the present technology, rather than to specify all permutations, combinations, and variations thereof in detail.

[0087] definition

[0088] In order to provide a clear and consistent understanding of the terms used in this specification, some definitions are provided below.In addition, unless otherwise defined, 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.

[0089] When used in conjunction with the term "comprising" in the claims and / or this specification, the use of the terms "a," "an," and "the" can mean "one," but it also complies with the following meanings: "one or more," "at least one," and "one or more than one." Similarly, the term "another" can mean at least a second or more. Unless otherwise specified herein or clearly contradicted by context, these terms should be construed to cover both the singular and the plural.

[0090] 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 nature 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 understood as closed.

[0091] The term "about" is used to indicate that a value or amount refers to the actual given value, as well as an approximate value of the given value that can be reasonably inferred based on ordinary skill in the art, including equivalent values ​​and approximate values ​​derived from the experimental and / or measurement conditions of the given value. For example, in the context of a given value or range, the term "about" 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.

[0092] When used herein, the expression "and / or" should be taken as a specific disclosure of each with or without a particular feature or component of the other. For example, "A and / or B" should be taken as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if listed individually herein. Unless otherwise specified or obvious from the context, the term "or" as used herein should be understood to be inclusive and encompass both "or" and "and". For example, an embodiment of "a composition comprising A or B" will generally present aspects of a composition comprising both A and B, however, "or" should be interpreted to exclude those presented aspects that cannot be combined without contradiction (e.g., a composition pH between 9 and 10 or between 7 and 8).

[0093] It should be understood herein that terms such as "1 to 20" include any individual value contained within (and including) 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 subrange contained within (and including) 1 to 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," and the like. 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," and the like.

[0094] It is understood herein that terms such as "about 15 to about 35" include any individual value subsumed between 15 and 35 (and including 15 and 35). Thus, terms such as "about 15 to about 35" include any number between 15 and 35 (and including 15 and 35), 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 subrange between 15 and 35 (and including 15 to 35), "about 16 to about 34," "about 16 to about 24," "about 24 to about 34," etc. In the context of an amino acid number, the term "about" is intended to specifically encompass the specified amino acid number and allow for a variation of + / - 2 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," "about 24 to about 34," etc.

[0095] It should be understood herein that terms such as "at least 80% identical" include any single value contained within 80% to 100% (and including 80% and 100%), 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 single sub-range contained within 80% to 100% (and including 80% to 100%), such as, for example, "85% to 99%", "97% to 100%", "90% to 100%", etc. The same applies to similar expressions, such as, but not limited to, "at least 70% identical", "at least 90% identical", etc.

[0096] 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 effects of varying concentrations of the inhibitor on the specific biological or biochemical function in question.

[0097] As used herein, the term "inhibitory efficacy" 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 cell receptor by its ligand). In some embodiments, the inhibitory efficacy is determined by measuring the IC50 of the inhibitor for a specific ligand or substrate. In this case, the relative inhibitory efficacy of different inhibitors and / or ligands can be assessed by comparing IC50 values. For example, a relative inhibitory efficacy of 3:1 means that the IC50 value ratio of the two substances compared is 3:1, wherein the first substance has a lower inhibitory efficacy (i.e., IC50 is greater) than the second substance. A relative inhibitory efficacy of 1:3 means that the IC50 value ratio of the two substances compared is 1:3, wherein the first substance has a greater inhibitory efficacy (i.e., IC50 is lower) than the second substance. Since the IC50 of an inhibitor can vary according to assay conditions, the relative inhibitory efficacy of different inhibitors and / or ligands is typically determined by comparing the IC50 values ​​obtained under the same assay conditions. The terms "inhibition potency," "inhibitory potency," "potency of inhibition," and "neutralization potency" are used interchangeably herein.

[0098] As used herein, the term "substantially identical" when referring to relative inhibitory potency means that two proteins have approximately the same relative inhibitory potency under the same experimental conditions, e.g., differ by no more than about 2-fold (+ / - 2-fold), e.g., the ratio of IC50 values of the two proteins is about 2: 1, 1:2, or 1:1.

[0099] As used herein, the term "functionally equivalent" means that a variant sequence 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 as compared to the original sequence. The term "substantially identical" means that a sequence is functionally equivalent to the original or reference sequence and has a high degree of sequence identity thereto. 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., hybridization and washing under salt and temperature conditions essentially equivalent to 0.5 X SSC to about 5 X SSC at 65 °C.

[0100] The term "dimer" refers to the presence of two polypeptides as described herein in a TGFβ superfamily ligand binding agent (also referred to herein as "binding agent"). "Homo-dimer" means that the two polypeptides have the same amino acid sequence, while "hetero-dimer" means that the two polypeptides have different amino acid sequences.

[0101] The term "bivalent" refers to the presence of two TGFβR superfamily ligand binding regions (e.g., ectodomains) in a TGFβ superfamily ligand binding agent.

[0102] As used herein, a "recombinant polypeptide" is a polypeptide made by the use of recombinant DNA technology or genetic engineering. In the context of the present disclosure, a recombinant polypeptide is often referred to as a "polypeptide construct" or simply as a "polypeptide."

[0103] Proteins (including fragments thereof, preferably biologically active fragments, and peptides, typically having less than 30 amino acids) comprise two or more amino acids coupled to each other via covalent peptide bonds (producing an amino acid chain). As used herein, the term "polypeptide" describes a group of molecules that are typically composed 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., consist of more than one polypeptide molecule. The polypeptide molecules that form such dimers, trimers, etc. can be identical or non-identical. Therefore, the corresponding higher-order structures of such multimers are referred to as homodimers or heterodimers, homotrimers, or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, the naturally occurring form of which 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, wherein the modification is achieved, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. The "peptides," "polypeptides," or "proteins" referred to herein may also be chemically modified, such as pegylated. Such modifications are well known in the art and are described herein.

[0104] As used herein, the terms "(specific) binding", "(specific) recognition", "specific for", "(specific) directed against" and "(specifically) react with" mean that a polypeptide interacts or specifically interacts with a given target, such as a specific member of the TGFβ ligand superfamily. It is believed that specific binding is influenced by specific motifs in the amino acid sequence of the polypeptide. Thus, binding is achieved as a result of their primary, secondary and / or tertiary structure, as well as as a result of secondary modifications of said structure. The specific interaction of a target interaction site with its specific target may result in simple binding of said site to the target. Furthermore, the specific interaction of a target interaction site with its specific target may alternatively or additionally result in the initiation of a signal, for example, due to the induction of a change in the conformation of the target, oligomerization of the target, etc., or may prevent the target from performing another activity, such as binding to an endogenous receptor.

[0105] In general, 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 or binds to a target can be readily tested, inter alia, by comparing the reaction of the polypeptide or binding agent with the target with the reaction of the polypeptide or binding agent with other proteins. In some embodiments, the polypeptides or binding agents of the present disclosure do not substantially bind to TGFβ superfamily ligands other than the desired ligand, for example, do not substantially bind to BMP-9.

[0106] As used herein, the term "does not substantially bind" or "is unable to bind" means that the polypeptide or binding agent of the present disclosure does not exhibit detectable binding to a given target, e.g., does not exhibit 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.

[0107] As used herein, the term "selectively binds" is used to refer to a polypeptide that binds to a target site that is not shared with other proteins. Generally, a selective binding agent does not cross-react with other proteins and only binds to the designated target protein. In the context of the present disclosure, "selective for activin A and GDF-8" means that the polypeptide or binding agent only binds to or neutralizes activin A and GDF-8 ligands, and does not substantially bind to or neutralize other TGFβ superfamily ligands (such as, for example, BMP-9).

[0108] "Half-life" refers to the time it takes for 50% of an administered drug to be eliminated through biological processes such as metabolism and excretion.

[0109] "First-pass hepatic metabolism" refers to the tendency of a drug to be metabolized during its first exposure to the liver, i.e., during its first pass through the liver.

[0110] "Volume of distribution" refers to the degree of retention of a drug in various body compartments, such as, for example, intracellular and extracellular spaces, tissues and organs, etc., and the distribution of the drug within these compartments.

[0111] "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.

[0112] The term "amino acid" or "amino acid residue" generally 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 (Ile 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 as desired. In general, amino acids can be grouped 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).

[0113] In a similar manner, "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 the candidate sequence that are identical to the nucleotide residues in the coding sequence of the polypeptide or binding agent. This is done using the BLASTN module of WU-BLAST-2 set to default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0114] TGFβ superfamily ligand binders

[0115] In some embodiments, the present disclosure provides a TGFβ superfamily ligand binding agent (also referred to herein as a "binding agent" or "TGFβ ligand binding agent") comprising an ActRIIB-ECD region, a linker region, and an Fc domain. The individual components of the binding agents described herein are described in further detail in the following sections. However, in general, the binding agents described herein are dimeric proteins comprising two polypeptides, each polypeptide comprising an ActRIIB-ECD, a peptide linker, and an Fc domain monomer. The two polypeptides are assembled via the Fc domain monomer to form the dimeric binding agent described herein. See Figure 8Schematic diagram in . 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 of the one or more ligands through their respective receptors, while not substantially binding to BMP-9 and / or BMP-10, and / or inhibiting signaling of BMP-9 and / or BMP-10 through their receptors. The binding agents can also have further biological activities or functions, such as binding to other ligands or targets, as further described herein.

[0116] In some embodiments, the binding agent of the present disclosure comprises two polypeptide chains, which are associated via the Fc domain monomer of an antibody or via a constant CH2 domain, a constant CH3 domain and / or via a combination of CH2 and CH3. The constant region of the antibody can be from a human IgG1, IgG2, IgG3 or IgG4 antibody, or substantially identical thereto. The association of the two polypeptide chains typically occurs during the expression and secretion of the protein, for example in mammalian cells. The Fc domain monomer typically comprises CH2, CH3, or CH2 and CH3 of an antibody heavy chain from human origin, and typically provides disulfide cross-linking between single-chain polypeptides. In one embodiment, the Fc domain monomer provides at least one disulfide bond between single-chain polypeptides. In another embodiment, the Fc domain monomer provides at least two disulfide bonds between single-chain polypeptides. In some cases, the antibody heavy chain also provides separation of dimeric polypeptides based on protein A, for example after production in a host cell.

[0117] As described above, point mutations in certain TGFβ superfamily ligand binders and ECDs are described in the art. See, for example, WO 2021 / 158675; WO 2022 / 150590; WO 2021 / 158675; WO 2022 / 072882; WO 2021 / 189019; and WO 2021 / 189010. Although point mutations in ActRIIB ECD have been described in the context of other TGFβ superfamily ligand binders, the effects of these mutations in the context of these previously described agents cannot predict the effects of these same mutations in the context of the binders described herein. See, for example, PCT / CA2023 / 050116, which describes the unpredictability of point mutations in ActRIIB ECD when combined with linkers of varying lengths. Therefore, the efficacy of the specific binding agents described herein depends not only on the mutations contained in the extracellular ligand binding domain, but also on the length of the linker used. As described herein, the length of the linker connecting the ActRIIB ECD and Fc domain has an unpredictable effect on the binding and inhibition of TGFβ superfamily ligands. Therefore, the present application provides TGFβ superfamily ligand binding agents that exhibit improved ligand binding characteristics and therapeutic efficacy. These compounds can be used to treat various diseases and conditions driven by TGFβ superfamily ligands, including pulmonary hypertension, muscle diseases, metabolic disorders, bone diseases, anemia, and fibrosis.

[0118] Other ECD-based capture agents, such as luspatercept and sotatercept, have been evaluated clinically. Luspatercept (also known as ACE-536 and REBLOZYL®) is a soluble fusion protein composed of a modified form of the extracellular domain of the activin type IIB receptor (ActRIIB) linked to the Fc portion of human IgG1. Luspatercept inhibits several endogenous TGFβ superfamily ligands, thereby attenuating Smad2 / 3 signaling. It is used to treat anemia in β-thalassemia and myelodysplastic syndrome. For descriptions of Rotacept 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.

[0119] Sotatercept (also known as ACE-011) is a soluble decoy receptor consisting of the extracellular domain of the activin type IIA receptor (ActRIIA) linked to the Fc portion of human IgGl and is capable of binding and neutralizing activin and GDFs. Sotatercept has been evaluated in healthy volunteers and patients with conditions characterized by dysfunction of TGF-beta superfamily signaling, including hematologic disorders, bone loss, chemotherapy-induced anemia, multiple myeloma, myelodysplastic syndrome, beta thalassemia, and end-stage renal disease (Raftopoulos, H., et al., 2016; Abdulkadyrov, K.M., et al., 2014; Ruckle, J., et al. 2009; Komrokji, R., et al., 2018; Cappellini, M.D., et al., 2019; Coyne, D.W., et al., 2019; Sherman, M.L., et al., 2013). Recently, sotatercept has been evaluated for the treatment of pulmonary arterial hypertension (PAH).

[0120] By acting as a ligand trap for activin and GDFs, soliatercept can correct the imbalance between the growth-promoting activin / growth differentiation factor pathway and the growth-inhibiting BMP pathway that occurs in PAH. In a Phase 2 trial in patients with PAH, soliatercept was shown to reduce pulmonary vascular resistance (Humbert, M. et al., 2021). Additional PH trials, including Phase 3 trials, are ongoing or planned. 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 for descriptions of soliatercept and other related fusion proteins. However, in some clinical studies of soliatercept, vascular and hematologic side effects were dose-limiting, limiting potential therapeutic efficacy. For example, a multiple ascending dose study in healthy postmenopausal women was designed to evaluate four doses of 0.1, 0.3, 1 mg / kg, and 2 mg / kg, but the study was terminated early at the 1 mg / kg level due to finding that increases in hemoglobin, hematocrit, and red blood cell count were dose-limiting (Sherman, M. L. et al., 2013). In a Phase 2 clinical trial in patients with PAH, thrombocytopenia and elevated hemoglobin levels were the most common hematologic adverse events, with 17% of patients receiving the 0.7 mg / kg dose experiencing an adverse event of elevated hemoglobin (Humbert, M. et al., 2021). These vascular and hematologic side effects are dose-limiting because they can not allow for administration of the dose required for maximum efficacy, limiting the potential to achieve maximum therapeutic effect (Humbert, M. et al., 2021). In contrast, the binding agents provided herein do not induce hematologic effects in non-human primates, suggesting that these drugs can have a wider therapeutic window than soliatercept.

[0121] In some embodiments, the binding agents of the present disclosure comprise a homodimer, i.e., a dimer of a polypeptide 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 a functional equivalent variant thereof. In other embodiments, the binding agents comprise a heterodimer, i.e., a dimer of two different polypeptides, 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 a functional equivalent variant thereof.

[0122] In some embodiments, the binding agents of the present disclosure comprise a dimer of a polypeptide comprising an ActRIIB-ECD comprising an F33Q mutation and a long peptide linker. In some embodiments, the linker is or is at least 10 amino acids in length. In some embodiments, the linker is or is at least 14 amino acids in length. In some embodiments, the linker is or is at least 19 amino acids in length. In some embodiments, the linker is or is at least 39 amino acids in length. In some embodiments, the binding agents of the present disclosure comprise 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 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 thereto, or a functionally equivalent variant thereof.

[0123] In some embodiments, the binding agents of the present disclosure comprise 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, the binding agents of the present disclosure comprise 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, the binding agents of the present disclosure comprise 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, the binding agents of the present disclosure comprise 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, the binding agents of the present disclosure comprise 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, the binding agents of the present disclosure comprise 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.

[0124] Activin type IIB receptor extracellular domain variant

[0125] As used herein, the term "activin type IIB receptor extracellular domain variant" or "ActRIIB-ECD variant" refers to a polypeptide comprising a soluble extracellular portion of a single transmembrane receptor ActRIIB that has at least one amino acid substitution relative to wild-type extracellular ActRIIB. The sequence of wild-type human ActRIIB-ECD is shown in SEQ ID NO: 2 (Table 1). Unless otherwise indicated, the indicated positions of amino acid substitutions are numbered according to the amino acid sequence of SEQ ID NO: 2. For the purposes of this disclosure, "human wild-type ActRIIB-ECD" refers to SEQ ID NO: 2.

[0126] In some embodiments, an ActRIIB-ECD variant comprises one or more amino acid substitutions at a position selected from the group consisting of L14, G27, L33, L55, and T69. In some embodiments, an ActRIIB-ECD variant polypeptide comprises one or more amino acid substitutions selected from the group consisting of 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 the group consisting of L33R, L33Y, L33F, L33Q, L33W, L33E, L33K, and L33M. In some embodiments, the ActRIIB-ECD variant comprises the amino acid substitution L33Y. In some embodiments, the ActRIIB-ECD variant comprises the amino acid substitution L33W. Other amino acid substitutions in ActRIIB-ECD are known in the art (e.g., WO 2021 / 158675; WO 2022 / 150590; WO 2021 / 158675; WO 2022 / 072882; WO 2021 / 189019; and WO 2021 / 189010, each of which is incorporated herein by reference). These additional mutations can be used in combination with the linkers described herein and incorporated into the binding agents described herein to alter the ligand binding properties of ActRIIB-ECD.

[0127] In some embodiments, the ActRIIB-ECD variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-62. In some embodiments, the ActRIIB-ECD variant has 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 greater) 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 greater) amino acid sequence identity to the sequence set forth in SEQ ID NO: 2.

[0128] In some embodiments, the amino acid sequence of the ActRIIB-ECD variant has 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: 4-62.

[0129] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 4-9. 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.

[0130] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 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.

[0131] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position T69 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to 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 SEQ ID NOs: 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.

[0132] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and position T69 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 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 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 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 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 positions L33Y and 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 positions L33Y and 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 positions L33Y and 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 positions L33F and T69Q and comprises or consists of the amino acid sequence of SEQ ID NO: 31.

[0133] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27 and position L33 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 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 SEQ ID NOs: 32-48. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position G27E and 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 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 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 positions G27Q and 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 positions G27K and 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 positions G27T and 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 positions G27M and 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 positions G27D and L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 39. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution 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 positions G27N and 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 positions G27Q and L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 42.In some embodiments, an ActRIIB-ECD variant comprises or consists of the amino acid sequence of SEQ ID NO: 43 comprising amino acid substitutions at positions G27D and L33W. In some embodiments, an ActRIIB-ECD variant comprises or consists of the amino acid sequence of SEQ ID NO: 44 comprising amino acid substitutions at positions G27E and L33W. In some embodiments, an ActRIIB-ECD variant comprises or consists of the amino acid sequence of SEQ ID NO: 45 comprising amino acid substitutions at positions G27N and L33W. In some embodiments, an ActRIIB-ECD variant comprises or consists of the amino acid sequence of SEQ ID NO: 46 comprising amino acid substitutions at positions G27Q and L33W. In some embodiments, an ActRIIB-ECD variant comprises or consists of the amino acid sequence of SEQ ID NO: 47 comprising amino acid substitutions at positions G27T and L33W. In some embodiments, an ActRIIB-ECD variant comprises or consists of the amino acid sequence of SEQ ID NO: 48 comprising amino acid substitutions at positions G27M and L33W.

[0134] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14 and position L33 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 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 SEQ ID NOs: 49-58. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14Q and L33Y and comprises or consists of the amino acid sequence of SEQ ID NO: 49. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L14D and 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 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 positions L14E and 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 positions L14H and 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 positions L14S and 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 positions L14E and 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 positions L14D and 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.

[0135] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and position L55 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 59-62. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33 and position L55 and comprises or consists of SEQ ID NOs: 59-62. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position L33Y and 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 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 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 positions L33Y and L55I and comprises or consists of the amino acid sequence of SEQ ID NO: 62.

[0136] Exemplary ActRIIB ECDs are provided in Table 1. Amino acid substitutions are indicated in bold and enlarged text.

[0137] Table 1: Exemplary ActRIIB and ActRIIB variant ECDs

[0138] In some embodiments, the ActRIIB-ECD variants of the present disclosure further include an extension of up to 5 amino acids at the N-terminus. In some embodiments, the ActRIIB-ECD variants of the present disclosure further include an extension of 5 amino acids at the N-terminus, such as GRGEA (SEQ ID NO: 63). In some embodiments, the ActRIIB-ECD variants of the present disclosure further include an extension of 4 amino acids, 3 amino acids, 2 amino acids, or 1 amino acid at the N-terminus, such as, but not limited to, RGEA, GEA, EA, or A. In some embodiments, the ActRIIB-ECD variants of the present disclosure further include an extension of 3 amino acids at the C-terminus. In some embodiments, the ActRIIB-ECD variants of the present disclosure further include an extension of 3 amino acids at the C-terminus, such as APT.

[0139] Table 2 provides exemplary ActRIIB ECDs with N-terminal and C-terminal extensions. The extended amino acids are indicated in bold and italic text. In some embodiments, any of SEQ ID NOs: 4-62 may further comprise an N-terminal or C-terminal extension.

[0140] Table 2: ActRIIB-ECD with N-terminal extension

[0141] ActRIIB-ECD variants of the present disclosure have been designed to maximize therapeutic efficacy in certain disease indications while minimizing adverse effects, specifically, to prevent or reduce interruption of endogenous BMP-9 and / or BMP-10 signaling while maintaining and / or increasing neutralizing potency for other TGFβ superfamily ligands such as activin A, activin B, GDF-8, and / or GDF-11. 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, which enables it 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, which avoids toxicity associated with inhibition of BMP-9 signaling; and optionally (3) similar or reduced binding to or inhibition of BMP-10, which avoids toxicity associated with inhibition of BMP-10 signaling. These variants can be used to treat a variety 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 miscellaneous PH), metabolic disorders and cardiometabolic diseases (e.g., obesity, type 1 diabetes, type 2 diabetes, prediabetes, heart failure), bone diseases (e.g., diseases or conditions involving bone injury), muscle diseases, fibrosis, and low red blood cell levels (e.g., anemia, blood loss), as described further herein. The variants can, for example but not limited to, result in reduction of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) symptoms or progression, reduction of bone resorption or osteoclast activity, increase in bone formation or bone mineral density, increase in muscle mass or strength, reduction in fibrosis (e.g., reduction in fibrosis or slowing or halting of fibrosis progression), and / or increase in red blood cell levels (e.g., increase in hemoglobin level, hematocrit, or red blood cell count, e.g., increase in red blood cell production), as described further herein.

[0142] In some embodiments, 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 by their respective receptors, without substantially binding to or inhibiting signaling by BMP-9 or BMP-10.

[0143] In some embodiments, ActRIIB-ECD variants of the present disclosure have about 5-fold reduced potency for inhibiting human BMP-9 signaling compared to the potency of human wild-type ActRIIB-ECD for inhibiting human BMP-9 signaling.

[0144] In some embodiments, the inhibitory potency of an ActRIIB-ECD variant of the present disclosure on human BMP-9 signaling is reduced by about 10-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD on human BMP-9 signaling.

[0145] In some embodiments, the inhibitory potency of an ActRIIB-ECD variant of the present disclosure on human BMP-9 signaling is reduced by about 100-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD on human BMP-9 signaling.

[0146] In some embodiments, the inhibitory potency of an ActRIIB-ECD variant of the present disclosure on human BMP-10 signaling is reduced by about 5-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD on human BMP-10 signaling.

[0147] In some embodiments, the inhibitory potency of an ActRIIB-ECD variant of the present disclosure on human BMP-10 signaling is reduced by about 10-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD on human BMP-10 signaling.

[0148] In some embodiments, the inhibitory potency of an ActRIIB-ECD variant of the present disclosure on human BMP-10 signaling is reduced by about 100-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD on human BMP-10 signaling.

[0149] In some embodiments, the ActRIIB-ECD variants of the present disclosure have inhibitory potency against one or more ligands selected from activin A, activin B, GDF-8, and GDF-11 that is substantially the same as or increased compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same one or more ligands.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] In some embodiments, compared to human wild-type ActRIIB-ECD, the ActRIIB-ECD variants of the present disclosure have higher inhibitory potency against activin A and / or activin B; lower inhibitory potency against BMP-9; and / or lower inhibitory potency against BMP-10.

[0157] 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.

[0158] Thus, in accordance with the present disclosure, provided herein are novel polypeptides comprising activin type IIB receptor (ActRIIB) extracellular domain (ECD) variants having one or more amino acid substitutions relative to the human wild-type ActRIIB-ECD sequence, with tailored TGFβ superfamily ligand specificity to maximize therapeutic efficacy while minimizing adverse effects, specifically with the goal of preventing or reducing disruption of endogenous BMP-9 and / or BMP-10 signaling while maintaining and / or increasing neutralization potency against other TGFβ superfamily ligands, such as activin A, activin B, GDF-8, and / or GDF-11.

[0159] Polypeptides comprising ActRIIB ECD variants

[0160] In some embodiments, the disclosure provides polypeptides 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 - a peptide linker - an Fc domain monomer. The polypeptides comprising an ActRIIB ECD can be dimerized via cysteine bonds between Fc domain monomers, forming a TGFβ superfamily ligand binding agent as described herein.

[0161] Linker

[0162] In some embodiments, the ActRIIB ECD variants described herein are 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 having one or more amino acid substitutions), an albumin binding peptide, a fibronectin domain, or a human serum albumin domain.

[0163] As used herein, the terms "peptide linker" and "linker" are used interchangeably to refer to a short segment of amino acids used to join two functional domains together in a polypeptide chain. For example, in some embodiments of the polypeptides or binding agents of the disclosure, the ActRIIB-ECD variant and the Fc domain monomer are joined together in a polypeptide chain via one or more peptide linkers. Peptide linkers can also be used to join other domains or modules or regions, such as a half-life extension domain, to the polypeptides or binding agents of the disclosure. As used herein, the term "long linker" refers to a linker that is at least 10 amino acids in length (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length). As used herein, the term "short linker" refers to a linker that is less than 10 amino acids in length (i.e., 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length).

[0164] 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 contain a motif, for example, a plurality of or repeated motifs 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).

[0165] In some embodiments, the linker can contain 2 to 12 amino acids comprising the motif GA or GS, for example, 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 contain 3 to 12 amino acids comprising the GGA or GGS motif, for example, 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 contain 4 to 12 amino acids comprising the motif 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 contain the motif GGGGA (SEQ ID NO: 124) or GGGGS (SEQ ID NO: 103), for example, GGGGAGGGGAGGGGA (SEQ ID NO: 151) and GGGGSGGGGSGGGGS (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 having 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).

[0166] In the case of using a joint, the length and sequence of the joint are generally sufficient to ensure that each domain can retain their difference binding specificity and / or function independently of each other. In some embodiments, the peptide joint that does not promote any secondary structure in addition is selected. The connection between the domains can be realized, for example, by genetic engineering as described herein. Preparation of fused and operably connected polypeptide constructs and expressing them in mammalian cells or bacteria is well known in the art (for example, WO 99 / 54440 or Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0167] In some embodiments, the joint includes various arrangements of amino acid sequences containing Gly and Ser. In some embodiments, the joint is a joint rich in glycine and serine. In some embodiments, the joint can be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or glycine and proline residues, and can for example comprise a single sequence of threonine / serine and glycine or a repetitive sequence of threonine / serine and / or glycine, such as 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) single sequence or repetitive sequence. Other near-neutral amino acids can also be used in the joint sequence, such as, but not limited to, Thr, Asn, Pro and Ala.

[0168] In some embodiments, the length of the linker is 10 amino acids. In some embodiments, the length of the linker exceeds 10 amino acids. In some embodiments, the length of 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 some embodiments, the linker is less than 40, 35, 30, 25, 22, or 20 amino acids. In some embodiments, the length of the linker is 10-50, 10-40, 10-30, 10-25, 10-21, 10-15, 10-14, 12-14, 15-25, 17-22, 20, or 21 amino acids. In some embodiments, the length of the linker is 14-40, 14-39, 14-35, 14-30, 14-25, or 14-20 amino acids. In some embodiments, the length of the linker is at least 10 amino acids. In some embodiments, the length of the linker is at least 14 amino acids. In some embodiments, the length of the linker is at least 19 amino acids. In some embodiments, the length of the linker is at least 39 amino acids. In some embodiments, the length of the linker is 14 amino acids. In some embodiments, the length of the linker is 19 amino acids. In some embodiments, the length of the linker is 39 amino acids. 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 is at least 12, 14, 15, 20, 21, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0169] In other embodiments, the length of the linker is less than 10 amino acids. In some embodiments, the length of the linker is 3 amino acids. In some embodiments, the length of the linker is 6 amino acids. In some embodiments, the length of the linker is 9 amino acids.

[0170] 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.

[0171] 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.

[0172] In some embodiments, the linker comprises or consists of a sequence set forth in any one of SEQ ID NOs: 68-170.

[0173] In some embodiments, the joint is a peptide rich in glycine, typically a peptide rich in glycine / serine, containing 40 amino acids at the most, or 1 to 40 amino acids, 2 to 39 amino acids, 3 to 39 amino acids, 3 to 14 amino acids, 3 to 19 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids or 15 to 25 amino acids. In some embodiments, the peptide linker only comprises the amino acid residues of relatively small number, for example, 39 amino acids or still less, 19 amino acids or still less, 14 amino acids or still less, 5 amino acids or still less, or 3 amino acids or still less. In certain embodiments, a joint rich in Gly is used. In one embodiment, the peptide linker can be made up of 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), wherein 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).

[0174] In some embodiments, the linker comprises the amino acid sequence GlyGlyGlyGlySer (GGGGS) (SEQ ID NO: 103), or repeats thereof (GGGGS)n, wherein n>2. In specific embodiments, n>3, or n=3-10. In some embodiments, n>4, or n=4-10. In some embodiments, n in the (GGGGS)n linker is no greater than 4. 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, n=4. In some embodiments, the linker comprising the (GGGGS)n sequence further comprises an N-terminal threonine.

[0175] In some embodiments, the linker can also contain 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), In some embodiments, the linker may contain a motif of a proline-rich sequence, such as a multiple or repeating motif, 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).

[0176] 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 required in the final protein fusion polypeptide. The length of the linker can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0177] Non-limiting examples of linkers are depicted in Table 3. It should be understood that the linker is not meant to be particularly limiting and any suitable linker can be used so long as it provides the desired function (binding, neutralizing, etc.) of the polypeptide or binding agent.

[0178] Table 3: Exemplary linker sequences

[0179] In some embodiments, ActRIIB ECD variant polypeptides or binding agents of the present disclosure comprise one or more linkers having a sequence set forth in any one of SEQ ID NOs: 89, 94, or 98. In some embodiments, ActRIIB ECD variant polypeptides or binding agents comprise a glycine-rich linker at the C-terminus of an ActRIIB ECD variant polypeptide, said linker being 2, 3, 6, 10, 14, 19, or 39 amino acids in length. In some embodiments, ActRIIB ECD variant polypeptides or binding agents of the present disclosure comprise a linker of SEQ ID NO: 89 at the C-terminus of an ActRIIB ECD variant polypeptide. In some embodiments, ActRIIB ECD variant polypeptides or binding agents of the present disclosure comprise a linker of SEQ ID NO: 94 at the C-terminus of an ActRIIB ECD variant polypeptide. In some embodiments, ActRIIB ECD variant polypeptides or binding agents of the present disclosure comprise a linker of SEQ ID NO: 98 at the C-terminus of an ActRIIB ECD variant polypeptide.

[0180] Fc domain monomer and Fc domain

[0181] 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. In some embodiments, the ActRIIB-ECD variant is fused at the C-terminus to the N-terminus of the Fc domain monomer via a linker.

[0182] As used herein, "Fc domain monomer" describes a single-chain protein that forms a functional Fc domain when associated with another Fc domain monomer. The association of two Fc domain monomers produces an Fc domain. As used herein, "Fc domain" describes the minimum region (in the case of a larger polypeptide) or minimum protein folding structure (in the case of an isolated protein) that can be bound to an Fc receptor (FcR) or bound by an Fc receptor. When two Fc domain monomers associate, the resulting Fc domain has Fc receptor binding activity. Therefore, the Fc domain is a dimeric structure that can bind to an Fc receptor. Unless otherwise indicated, reference to "variant Fc domain" herein should be understood to refer to a dimeric Fc domain, wherein each Fc domain monomer includes the mutation mentioned.

[0183] It should be understood that the Fc domains as used herein include polypeptides comprising antibody constant regions (excluding the first constant region immunoglobulin domain). Therefore, Fc refers to the last two constant region immunoglobulin domains (CH2, CH3) of IgG and the flexible hinge at the N-terminal ends of these domains. Although the boundaries of the Fc domain monomers may be different, human IgG heavy chain Fc domain monomers are generally defined as comprising residues C226 or P230 at their carboxyl termini. Unless otherwise indicated, the amino acid positions in all Fc domains and Fc domain monomers mentioned are based on the EU index listed in Kabat (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). Fc can refer to this region alone, or to this region 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, so there may be subtle differences between the sequences provided herein and those in the art. The Fc domain monomer comprised in the polypeptides or binding agents of the present disclosure may be an IgG1, IgG2, IgG3 or IgG4 domain.

[0184] In exemplary embodiments, the polypeptide of the present disclosure comprises one or more constant regions of an antibody, such as 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 can be, for example, but not limited to, an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody. In specific embodiments, the antibody is a human antibody, for example, and the Fc domain monomer comprises the constant region of the heavy chain of human IgG1, IgG2, IgG3 or IgG4. 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 with human IgG1, IgG2, IgG3 or IgG4 constant region. In specific embodiments, the Fc domain monomer comprises or consists of the Fc domain monomer of a human IgG1 antibody. In another specific embodiment, the Fc domain monomer comprises or consists of the Fc domain monomer of a human IgG2 antibody. In another specific embodiment, the Fc domain monomer comprises or consists of the Fc domain monomer of a human IgG4 antibody. Exemplary Fc domain sequences (including wild-type sequences, polymorphic and variant sequences thereof) are provided in Table 4.

[0185] Table 4: Exemplary Fc domain sequences

[0186] In some embodiments, the IgG1 sequences of the present disclosure further include a 2 amino acid extension at the N-terminus, such as DK.

[0187] Generally, the ActRIIB-ECD polypeptide is organized such that the Fc domain monomer is linked at its N-terminus to the C-terminus of the ActRIIB-ECD variant, such that for each ActRIIB-ECD polypeptide, the construct is oriented from N-terminus to C-terminus as a single chain (ActRIIB-ECD variant)-(linker)-(Fc domain monomer). However, the orientation of the construct is not particularly limited, and other orientations are contemplated. For example, in some embodiments, the Fc domain monomer can be linked at its C-terminus to the N-terminus of the ActRIIB-ECD variant.

[0188] In an exemplary embodiment, the Fc domain monomer allows two or more polypeptide chains to be assembled in a covalent manner, for example, by disulfide bonds between cysteine ​​residues. In this way, the Fc domain monomer acts as a dimerization domain, allowing two ActRIIB-ECD polypeptide chains to assemble to form a dimer. According to the present disclosure, such dimers typically comprise two polypeptides, each comprising an ActIIRB-ECD variant connected to an Fc domain monomer as described herein, thereby forming a bivalent TGFβ superfamily ligand binding agent. Therefore, the binding agent described herein comprises two ActIIRB-ECD variants, a linker domain, and an Fc domain.

[0189] Fc domain monomers generally include one or more cysteine ​​residues for cross-linking the first polypeptide to the second polypeptide in a homodimer construct. For example, an Fc domain monomer may include at least two cysteine ​​residues for forming a disulfide bridge between two polypeptides to form a dimer. In some embodiments of the present technology, an Fc domain monomer includes or is composed of: SEQ ID NO: The sequence set forth in any one of 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 a specific embodiment, an Fc domain monomer includes SEQ ID NO: The amino acid sequence set forth in 253 or is composed thereof. In a specific embodiment, an Fc domain monomer includes SEQ ID NO: The amino acid sequence set forth in 266 or is composed thereof. In a specific embodiment, an Fc domain monomer includes SEQ ID NO: The amino acid sequence set forth in 256 or is composed thereof. In a specific embodiment, an Fc domain monomer includes SEQ ID NO: The amino acid sequence set forth in 255 or is composed thereof.

[0190] In some embodiments, the present disclosure provides binding agents comprising a variant Fc domain (ie, a non-naturally occurring Fc domain, eg, an Fc domain comprising one or more non-naturally occurring amino acid residues, substitutions, additions, deletions, etc.).

[0191] 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 desired properties (such as increased half-life) compared to a naturally occurring (wild-type) Fc sequence. As used herein, a "variant Fc domain" refers to a non-naturally occurring Fc domain, e.g., an Fc domain comprising 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.

[0192] There are many known polymorphs of IgG1 Fc domains, including the "DEL" polymorph and the "EEM" polymorph. The DEL polymorph comprises 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 comprises 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 Fc domain or an EEM Fc domain are expected to exhibit similar properties with respect to 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, such as the IgG1 polymorphs of SEQ ID NOs: 252-253 and 258-266, the IgG2 polymorphs of SEQ ID NOs: 267-276, the IgG3 polymorphs of SEQ ID NOs: 277-283, and the IgG4 polymorphs of SEQ ID NOs: 284-292.

[0193] In some embodiments, the variant Fc domain formed by two variant Fc domain monomers has a changed binding property to Fc receptors (such as FcRn) relative to comparable molecules (for example, proteins with the same amino acid sequence except for wild-type Fc domain monomers). The serum half-life of the protein comprising the 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 increased serum half-life relative to comparable molecules. In a specific embodiment, the Fc domain variant comprises at least one amino acid substitution (referred to herein as "YTE"; for example, SEQ ID NO: 254 and 255) selected from the group consisting of M252Y, S254T and T256 at one or more positions. In another embodiment, the Fc domain variant comprises Y (for example, SEQ ID NO: 256 and 257, referred to herein as "Fc-Y") at position 252. In another embodiment, the Fc domain variant comprises T at position 254. In another embodiment, the Fc domain variant comprises E at position 256.

[0194] 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. In 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.

[0195] 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 substitutions, such as, but not limited to, E356D and M358L.

[0196] In some embodiments, an 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 a DEL polymorph (referred to herein as YTE-DL, e.g., SEQ ID NO: 255). In some embodiments, the FcYTE domain monomer is an EEM polymorph (referred to herein as YTE-EM, e.g., SEQ ID NO: 254).

[0197] In some embodiments, an 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 a DEL polymorph (referred to herein as Y-DL, e.g., SEQ ID NO: 256). In some embodiments, the FcY domain monomer is an EEM polymorph (referred to herein as Y-EM, e.g., SEQ ID NO: 257).

[0198] In some embodiments, the ActRIIB-ECD polypeptide comprises an Fc domain monomer containing a lysine residue (K) at the C-terminus.

[0199] In some embodiments, variant Fc domains (e.g., Fc domains formed by two variant Fc domain monomers) for use in the ActRIIB-ECD polypeptides of the present disclosure comprise one or more amino acid substitutions that reduce ActRIIB-ECD polypeptide aggregation and / or increase stability and / or increase half-life compared to naturally occurring Fc sequences. In some embodiments, the Fc domains are selected to provide one or more effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation (complement-dependent cytotoxicity or CDC), opsonization, and the like. In one embodiment, the variant Fc domains have enhanced binding to Fc receptors relative to comparable molecules. In specific embodiments, the variant Fc domains have enhanced binding to the neonatal Fc receptor, FcRn. In another embodiment, the variant Fc domain and / or polypeptide or binding agent comprising 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 increased serum half-life relative to a comparable molecule.

[0200] Examples of methods for extending the serum half-life of polypeptides and binding agents of the present invention include peptides, proteins or protein domains fused to or otherwise connected to polypeptides and binding agents. Peptide, protein or protein domain groups include peptides that bind to other proteins with preferred pharmacokinetic characteristics in humans, such as serum albumin (see WO 2009 / 127691). Alternative concepts for such peptides that extend the half-life include peptides that bind to neonatal Fc receptors (FcRn, see WO 2007 / 098420), which can also be used in polypeptides and binding agents of the present invention. The concept of linking larger protein domains or intact proteins includes, for example, human serum albumin, human serum albumin variants or mutants (see WO 2011 / 051489, WO 2012 / 059486, WO 2012 / 150319, WO 2013 / 135896, WO 2014 / 072481, WO 2013 / 075066) or fusions of their domains and fusions of immunoglobulin constant regions (Fc domains) and variants thereof, as described herein. Such Fc domain variants can be optimized / modified to allow the desired pairing of dimers or multimers, to eliminate Fc receptor binding (e.g., Fcg receptors), to enhance binding to FcRn, or for other reasons. Another concept known in the art to extend the half-life of small protein compounds in humans is to pegylate these compounds (such as the polypeptides or binding agents of the present disclosure).

[0201] In one embodiment, the present disclosure provides a binding agent wherein the Fc domain comprises a non-naturally occurring amino acid residue at one or more positions 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, 279, 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 are numbered according to the EU index as set forth in Kabat. Optionally, the Fc domain may comprise 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 Publications WO 01 / 58957, WO 02 / 06919, WO 04 / 016750, WO 04 / 029207, WO 04 / 035752, WO 04 / 074455, WO 04 / 099249, WO 04 / 063351, WO 05 / 070963, WO 05 / 040217, WO 05 / 092925, and WO 06 / 020114). In specific embodiments, the present disclosure provides Fc variant protein compositions, wherein the Fc domain comprises at least one amino acid substitution selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R.243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263 1. 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 26 5I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 2 92L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325 H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 32 9Q, 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 are numbered according to the EU index as set forth in Kabat. Optionally, the Fc domain may comprise 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 Publications WO 01 / 58957, WO 02 / 06919, WO 04 / 016750, WO 04 / 029207, WO 04 / 035752, and WO 05 / 040217).

[0202] Another domain

[0203] It is contemplated that the ActRIIB-ECD polypeptides and / or binding agents of the present disclosure can have further binding specificities or further functions in addition to the function of binding to a target TGFβ superfamily ligand 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 can have further functions, such as a fully functional Fc constant domain that mediates antibody-dependent cellular cytotoxicity by recruiting effector cells (e.g., NK cells), by providing a label (fluorescence, etc.), by providing a therapeutic agent (such as a toxin or a radionuclide), and / or by providing a means to enhance serum half-life, etc.

[0204] In some embodiments, the ActRIIB-ECD polypeptides described herein comprise an 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, 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, for example, transmembrane receptor proteins (such as integrins) and extracellular matrix components (such as collagens and fibrillins). In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the UniProt ID NO: P02751 sequence. In other embodiments, the fibronectin domain is an adnectin protein.

[0205] In some embodiments, polypeptides or binding agents of the present disclosure include ActRIIB-ECD variants fused to one or more fibronectin domains. Combining with fibronectin domains can improve the pharmacokinetics of protein drugs. Fibronectin domains refer to high molecular weight glycoproteins or fragments thereof of the extracellular matrix, which are bound to, for example, transmembrane receptor proteins (such as integrins) and extracellular matrix components (such as collagen and fibrin). In some embodiments of the present invention, fibronectin domains are joined to ActRIIB-ECD variants as described herein (e.g., ActRIIB-ECD variants with SEQ ID NO: 4-62 amino acid sequences set forth in any one) N-terminal or C-terminal (e.g., C-terminal) to increase the serum half-life of ActRIIB-ECD variants. Fibronectin domains can be joined directly or via a linker to the N-terminal or C-terminal of an ActRIIB-ECD variant or its polypeptide or its binding agent. In some embodiments, polypeptides or binding agents of the present disclosure can be fused to the N-terminal or C-terminal of a fibronectin domain, for example, by conventional genetic or chemical means (e.g., 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 prolonged retention of the therapeutic protein through its binding to integrins and extracellular matrix components (such as collagen and fibrin).

[0206] As an example, fibronectin domains that can be used in the methods, compositions, and polypeptides of the present disclosure are well 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 ID NO: P02751. In another embodiment, the fibronectin domain is an adnectin protein.

[0207] 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 accounts for approximately half of serum protein. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768.

[0208] In some embodiments, the ActRIIB variants or polypeptides or binding agents described herein can be fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of protein drugs. Serum albumin is a globular protein and the most abundant blood protein in mammals. Serum albumin is produced in the liver and accounts for about half of serum protein. 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 required for the proper distribution of body fluids between blood vessels and body tissues. In some embodiments, serum albumin is human serum albumin. In some embodiments, human serum albumin is joined 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 joined to the N-terminus or C-terminus of an ActRIIB-ECD variant directly or via a linker.

[0209] As an example, serum albumin that can be used in the polypeptides and methods and compositions described herein is well known in the art. In one embodiment, serum albumin comprises the sequence of UniProt ID NO: 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, by conventional genetic or chemical means (e.g., chemical conjugation). If desired, a linker (e.g., a spacer) can be inserted between the ActRIIB-ECD variant and the 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 extended retention of the therapeutic protein.

[0210] In some embodiments, the polypeptides or binding agents of the present disclosure further comprise 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 amino acid substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) that can be fused to the N-terminus or C-terminus (e.g., 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 can form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers that bind to form the Fc domain in the dimer. Furthermore, in some embodiments, the polypeptides or binding agents described herein have a serum half-life in humans of at least 7 days.

[0211] Exemplary TGFβ Superfamily Binding Agents

[0212] The overall structures of exemplary binding agents described herein are provided in Table 5. The amino acid sequence of each binding agent is provided in Table 6.

[0213] Table 5: Structures of exemplary binding agents

[0214] Table 6: Exemplary binding agent amino acid sequences Bold and italic text indicates linker sequences; bold text indicates N-terminal extension amino acids In some embodiments, the binding agent comprises an ActRIIB ECD, a peptide linker, and an Fc domain from N-terminus to C-terminus. 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 positions selected from L14, G27, L33, L55, and L69, wherein the amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the amino acid 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 amino acid substitution at position T69 is selected from T69H, T69Q, T69E, T69R, T69Y and T69W.

[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 substitution G27E; a peptide linker of 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: 4; a peptide linker of 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: 4; a peptide linker of 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 that is 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).

[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 substitution L33R; a peptide linker of 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: 15; a peptide linker of 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: 15; a peptide linker of 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 that is 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).

[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 the amino acid substitution L33Y; a peptide linker of 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: 12; a peptide linker of 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: 12; a peptide linker of 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 that is 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).

[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 substitution T69H; a peptide linker of 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: 19; a peptide linker of 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: 19; a peptide linker of 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 that is 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).

[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 substitution T69Q; a peptide linker of 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: 20; a peptide linker of 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: 20; a peptide linker of 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 that is 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).

[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 the amino acid substitution G27E; a peptide linker of 3 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: 4; a peptide linker of 3 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: 4; a peptide linker of 3 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 that is 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).

[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 substitution L33Y; a peptide linker of 3 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: 12; a peptide linker of 3 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: 12; a peptide linker of 3 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 that is 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).

[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 substitution T69H; a peptide linker of 3 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: 19; a peptide linker of 3 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: 19; a peptide linker of 3 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 that is 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).

[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 substitution T69Q; a peptide linker of 3 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: 20; a peptide linker of 3 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: 20; a peptide linker of 3 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 that is 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).

[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 amino acid substitutions L33Y and T69H; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 that is 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).

[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 amino acid substitutions L33Y and T69Q; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 or consists of the amino acid sequence of 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).

[0226] 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 amino acid substitutions L33Y and T69E; a 3aa, 10aa, or 14aa long peptide linker; and an IgGl 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30; a 3aa, 10aa, or 14aa long peptide linker; and an IgGl-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 3aa, 10aa, or 14aa long peptide linker; and an IgGl-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 that is 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 the amino acid sequence of or consists 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 that is 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 the amino acid sequence of or consists 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 that is 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 the amino acid sequence of or consists of SEQ ID NO: 191 (P1179).

[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 amino acid substitutions L33Y and G27D; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 that is 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).

[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 amino acid substitutions L33Y and G27E; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 that is 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).

[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 amino acid substitutions L33F and T69Q; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 that is 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).

[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 the amino acid substitution T69E; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 that is 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).

[0231] 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 amino acid substitution L33F; a 3aa, 10aa, or 14aa long peptide linker; and an IgGl 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10; a 3aa, 10aa, or 14aa long peptide linker; and an IgGl-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 3aa, 10aa, or 14aa long peptide linker; and an IgGl-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 that is 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 the amino acid sequence of or consists 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 that is 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 the amino acid sequence of or consists 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 that is 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 the amino acid sequence of or consists of SEQ ID NO: 206 (P1206).

[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 amino acid substitution L33Q; a peptide linker of 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 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, from N-terminus 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 that is 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 that is 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 that is 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).

[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 substitution L33Y; a peptide linker of 10 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: 12; a peptide linker of 10 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: 12; a peptide linker of 10 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 that is 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).

[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 substitution L33W; a peptide linker of 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: 13; a peptide linker of 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: 13; a peptide linker of 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 that is 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).

[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 substitution L33E; a peptide linker of 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: 16; a peptide linker of 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: 16; a peptide linker of 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 that is 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).

[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 substitution L33K; a peptide linker of 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: 17; a peptide linker of 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: 17; a peptide linker of 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 that is 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).

[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 substitution L33M; a peptide linker of 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: 18; a peptide linker of 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: 18; a peptide linker of 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 that is 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).

[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 amino acid substitutions L33Y and G27N; a peptide linker of 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 of 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 of 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 that is 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).

[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 amino acid substitutions L33Y and G27Q; a peptide linker of 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 of 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 of 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 that is 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).

[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 amino acid substitutions L33Y and G27K; a peptide linker of 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 of 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 of 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 that is 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).

[0241] 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 amino acid substitutions L33Y and G27Y; a 14 aa long peptide linker; and an IgGl 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37; a 14 aa long peptide linker; and an IgGl-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: 37; a 14 aa long peptide linker; and an IgGl-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 that is 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).

[0242] 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 amino acid substitutions L33Y and G27M; a 14 aa long peptide linker; and an IgGl 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38; a 14 aa long peptide linker; and an IgGl-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: 38; a 14 aa long peptide linker; and an IgGl-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 that is 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).

[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 amino acid substitutions L33Y and T69R; a peptide linker of 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 of 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 of 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 that is 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).

[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 amino acid substitutions L33Y and T69Y; a peptide linker of 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: 26; a peptide linker of 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: 26; a peptide linker of 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 that is 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).

[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 amino acid substitutions L33Y and T69W; a peptide linker of 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 of 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 of 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 that is 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).

[0246] 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 amino acid substitutions L33Y and L14E; a 14 aa in length peptide linker; and an IgGl 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52; a 14 aa in length peptide linker; and an IgGl-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: 52; a 14 aa in length peptide linker; and an IgGl-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 that is 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).

[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 amino acid substitutions L33Y and L14H; a peptide linker of 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 of 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 of 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 that is 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).

[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 amino acid substitutions L33Y and L14S; a peptide linker of 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 of 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 of 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 that is 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).

[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 amino acid substitutions L33Y and L55Y; a peptide linker of 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: 59; a peptide linker of 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: 59; a peptide linker of 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 that is 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).

[0250] 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 amino acid substitutions L33Y and L55Q; a peptide linker of 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 of 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 of 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 that is 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).

[0251] 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 amino acid substitutions L33Y and L55M; a peptide linker of 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 of 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 of 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 that is 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).

[0252] 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 amino acid substitutions L33Y and L55I; a peptide linker of 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 of 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 of 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 that is 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).

[0253] 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 substitution L33W; a peptide linker of 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: 13; a peptide linker of 14 aa in length; and an IgG1 γ-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 of 14 aa in length; and an IgG1 γ-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 that is 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).

[0254] 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 amino acid substitution L33W; a 14 aa in length peptide linker; and an IgGl 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; a 14 aa in length peptide linker; and an IgGl 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- to C-terminus: an ActRIIB ECD comprising or consisting of the amino acid sequence of SEQ ID NO: 13; a 14 aa in length peptide linker; and an IgGl 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 that is 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).

[0255] 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 substitution L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0256] 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 substitution L33W; a peptide linker of 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 of 19 aa in length; and an IgG1 γ-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 of 19 aa in length; and an IgG1 γ-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 that is 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).

[0257] 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 substitution L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0258] 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 amino acid substitutions G27D and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0259] 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 amino acid substitutions G27E and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0260] 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 amino acid substitutions G27N and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0261] 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 amino acid substitutions G27Q and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0262] 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 amino acid substitutions G27T and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0263] 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 amino acid substitutions G27M and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0264] 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 amino acid substitutions G27D and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0265] 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 amino acid substitutions G27E and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0266] 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 amino acid substitutions G27N and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0267] 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 amino acid substitutions G27Q and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0268] 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 amino acid substitutions L14E and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0269] 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 amino acid substitutions L14D and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0270] 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 amino acid substitutions L14N and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0271] 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 amino acid substitutions L14Q and L33W; a peptide linker of 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 of 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 of 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 that is 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).

[0272] 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 amino acid substitutions L14Q and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0273] 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 amino acid substitutions L14D and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0274] 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 amino acid substitutions L14N and L33Y; a peptide linker of 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 of 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 of 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 that is 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).

[0275] In some embodiments, the polypeptides or binding agents disclosed herein are "isolated" or "substantially pure." When used to describe the polypeptides or binding agents disclosed herein, "isolated" or "substantially pure" means 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 from or substantially free from association with all other components from its production environment. Contaminant components in its production environment (such as those produced by recombinant transfected cells) are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The polypeptide or binding agent may, for example, comprise at least about 5% or at least about 50% (by weight) of the total protein in a given sample. It should be understood that, depending on the circumstances, the isolated protein may comprise from 5% to 99.9% (by weight) of the total protein content. Significantly higher concentrations of the polypeptide or binding agent can be prepared by using an inducible promoter or a high-expression promoter, thereby preparing the polypeptide or binding agent at increased concentration levels. This definition includes production of the polypeptide or binding agent in various organisms and / or host cells known in the art. In preferred embodiments, the polypeptide or binding agent will be purified (1) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. However, typically, the isolated polypeptide or binding agent will be prepared by at least one purification step, such as, for example, but not limited to, affinity chromatography and / or ion exchange chromatography, e.g., binding to a Protein A column.

[0276] In some embodiments, the polypeptides and binding agents of the present disclosure are characterized by, for example, one or more of the following: particularly high affinity for one or more of activin A, activin B, GDF-8, and GDF-11; high neutralizing potency (low IC50 value) 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 value) 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.

[0277] The biological activity of the polypeptide or binding agent of the present invention or its pharmaceutical composition can be determined by, for example, cell neutralization assays, binding assays, competition assays, etc. As used herein, "efficacy" or "in vivo efficacy" refers to the response to a therapy using the polypeptide or binding agent or pharmaceutical composition of the present invention. The success or in vivo efficacy of a therapy using the polypeptide or binding agent or pharmaceutical composition of the present invention refers to the effectiveness of the polypeptide or binding agent or composition for its intended purpose, for example, the ability of the polypeptide or binding agent or composition to cause its desired effect (i.e., to treat, improve or prevent a TGFβ superfamily-related disease or condition as defined herein). In vivo efficacy can be monitored by established standard methods for the respective disease entities. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used.

[0278] Another major challenge in developing drugs, such as the pharmaceutical compositions of the present disclosure, is the predictable regulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of a drug candidate can be established, i.e., a profile of pharmacokinetic parameters that influence the ability of a particular drug to treat a particular condition. Pharmacokinetic parameters that influence the ability of a drug to treat a particular disease entity include, but are not limited to, half-life, volume of distribution, first-pass metabolism in the liver, and serum binding. The efficacy of a given drug can be affected by each of these parameters.

[0279] Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rate, multiple onset and / or Cmax of a given dose of a drug. "Bioavailability" refers to the amount of a drug in the blood compartment. "Lag time" refers to the time delay between drug administration and the detection and measurable presence of the drug in the blood or plasma. "Tmax" is the time it takes for a drug to reach maximum blood concentration, while "Cmax" is the maximum blood concentration achieved for a given drug. The time it takes for a drug to reach the blood or tissue concentration required for its biological effect is affected by all of these parameters.

[0280] In some embodiments, the half-life of a polypeptide or binding agent of the disclosure is about 3 days or longer, about 5 days or longer, about 1 week or longer, about 2 weeks or longer, about 3 weeks or longer, about 4 weeks or longer, about 5 weeks or longer, about 6 weeks or longer, or about 2 months or longer.

[0281] In some embodiments, the polypeptides or binding agents of the present disclosure may exhibit favorable thermal stability, with an aggregation temperature of about 45°C or higher, about 45°C to about 50°C, about 52°C to about 54°C, about 56°C to about 60°C, or about 60°C or higher. The thermal stability parameter can be determined based on the aggregation temperature of the polypeptide as follows: a solution of the protein at a test concentration (e.g., 100 μg / ml, 250 μg / ml) is transferred to a disposable cuvette and placed in a dynamic light scattering (DLS) apparatus. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min while the measured radius is continuously acquired. An increase in radius indicates protein melting and aggregation, which is used to calculate the aggregation temperature of the polypeptide. Other methods known in the art can be used.

[0282] In one embodiment, the polypeptide or binding agent according to the present disclosure is stable at 2°C-8°C for at least 1 month, 2 months or 3 months. In one embodiment, the polypeptide or binding agent according to the present disclosure is stable at 25°C-40°C for at least 4 weeks. In one embodiment, the polypeptide or binding agent according to the present disclosure is stable after undergoing 3 freeze / thaw cycles. In one embodiment, the polypeptide or binding agent according to the present disclosure is stable at -20°C for 1 month, 2 months, 3 months or longer.

[0283] Alternatively, the intrinsic biophysical protein stability of a polypeptide or binding agent can be determined by determining a temperature melting curve by differential scanning calorimetry (DSC). These experiments can be performed using a MicroCal LLC (Northampton, Mass., USA) VP-DSC apparatus. The energy absorption of a sample containing the polypeptide or binding agent compared to a sample containing formulation buffer alone is recorded from 20°C to 90°C. To record the corresponding melting curve, the overall sample temperature is increased stepwise. At each temperature T, the energy absorption of the sample and the formulation buffer reference is recorded. The difference in energy absorption Cp (kcal / mole / °C) of the sample minus the reference is plotted against the corresponding temperature. The melting temperature is defined as the temperature at which the energy absorption first reaches a maximum.

[0284] In another embodiment, 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 a non-physiological pH such as pH 5.5 (the pH required for running, for example, 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.

[0285] Amino acid sequence modification

[0286] Amino acid sequence modifications 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 polypeptide or binding agent. Amino acid sequence modifications / variants of polypeptides and binding agents are typically prepared by introducing appropriate nucleotide changes in the encoding nucleic acid or by peptide synthesis. All of the following amino acid sequence modifications should produce polypeptides or binding agents that still retain the desired biological activity of the unmodified parent molecule (e.g., binding to one or more of activin A, activin B, GDF-8, GDF-11, but substantially not binding to BMP-9 and BMP-10).

[0287] As used herein, the term "functional equivalent" refers to that the modified sequence has the same or substantially the same biological activity or function as the original sequence from which it is derived, such as, compared to the original sequence, there is no significant change in physiology, chemistry, physicochemistry or functional properties. The term "substantially identical" refers to that a sequence is functionally equivalent to an original or reference sequence and has a high degree of sequence identity therewith. In general, substantially identical sequences are 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 have the same function. In some cases, when referring to a nucleic acid sequence, substantially identical sequences hybridize with the original sequence under high stringency conditions, such as, hybridize and wash under conditions of salt and temperature that are substantially equivalent to 0.5 × SSC to about 5 × SSC and 65°C. In general, it is intended to encompass sequences that are substantially identical or functionally equivalent to a sequence provided according to the present disclosure.

[0288] Amino acid modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of a polypeptide or binding agent. Any combination of deletions, insertions, and substitutions is performed to obtain the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processes 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 alter glycosylation sites.

[0289] For example, one, two, three, four, five, or six amino acids may be inserted or deleted in a polypeptide or binding agent. Preferably, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one, two, three, four, five, six, seven, eight, nine, or ten residues to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Insertion variants of the disclosed polypeptides or binding agents include fusions to the N- or C-termini of enzyme polypeptides or binding agents, or to polypeptides that increase the serum half-life of the polypeptide or binding agent.

[0290] Modifications of the polypeptides or binding agents described herein are also contemplated. Modifications encompassed by the present disclosure include modifications having alterations in the amino acid sequence of the polypeptide or binding agent. Modifications of the polypeptide or binding agent include, for example, modifications that have similar or improved binding affinity, avidity, ligand specificity, inhibitory potency, stability, manufacturability, half-life, and / or reduced aggregation compared to the polypeptides or binding agents disclosed herein.

[0291] A target site of substitution mutagenesis includes an Fc domain monomer as described above. Exemplary embodiments of the modified polypeptide or binding agent of the present disclosure may include a polypeptide or binding agent with a modified IgG1, IgG2, IgG3 or IgG4 constant region or a portion 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).

[0292] Modifications encompassed by the present disclosure include those that may comprise insertions, deletions, or amino acid substitutions (conservative or non-conservative). These modifications may remove at least one amino acid residue in its amino acid sequence and insert a different residue in its place. It should be understood that alterations may occur in multiple regions of a polypeptide or binding agent as long as the desired binding or biological activity is maintained.

[0293] It is known in the art that modifications and variants can be generated by substitution mutagenesis while retaining the biological activity (i.e., functional equivalence) of the polypeptides disclosed herein. These modifications or variants remove at least one amino acid residue in the amino acid sequence and insert a different residue in its place, such as one or more conservative amino acid substitutions. In general, conservative amino acid substitutions refer to the substitution of one amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).

[0294] Generally, the degree of similarity and identity between variant polypeptide chains was determined 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; gapx dropoff 50, expectation 10.0, word length 3) and activation filters.

[0295] However, the level of identity can also be determined over the entire length of a given sequence. Thus, the percent identity will indicate the amino acids that are identical and that may occupy the same or similar positions compared to the original peptide. The percent similarity will indicate the identical amino acids at the same or similar positions compared to the original peptide and the amino acids that have been substituted using conservative amino acid substitutions.

[0296] Thus, in some embodiments, modifications of the polypeptides or binding agents of the present disclosure 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.

[0297] In some embodiments, the substitution is a conservative substitution. However, any substitution (including non-conservative substitutions) is contemplated as long as the polypeptide or binding agent retains its ability to bind and / or inhibit the desired TGFβ superfamily ligand without substantially binding to or inhibiting BMP-9 and / or BMP-10.

[0298] Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequences encoding the polypeptides or binding agents of the present disclosure and the nucleotide sequences described herein is at least 60%, and more typically, preferably 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 nearly 100%.

[0299] Nucleic acid and peptide and binding agent production

[0300] The present disclosure also provides polynucleotides encoding ActRIIB-ECD polypeptides provided herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence that is 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 polynucleotide encodes an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 174-251. In some embodiments, the polynucleotide encodes an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 174-251.

[0301] Polynucleotides are biopolymers composed of nucleotide monomers covalently bonded to a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotides with different biological functions. Nucleotides are organic molecules that serve as monomers or subunits of nucleic acid molecules such as DNA or RNA. Nucleic acid molecules or polynucleotides can be double-stranded or single-stranded, linear or circular. In some embodiments, the nucleic acid molecules or polynucleotides are contained in a vector. In some embodiments, the vector is contained in a host cell. The host cell can express a polypeptide or binding agent, for example, after transformation or transfection with the vector or polynucleotide of the present disclosure. For this purpose, the polynucleotide or nucleic acid molecule is typically operably linked to a control sequence.

[0302] Furthermore, the present disclosure provides vectors comprising a polynucleotide / nucleic acid molecule encoding a polypeptide or binding agent provided herein.

[0303] Vector is a nucleic acid molecule used as a vehicle for transferring (external) genetic material into cells. The term "vector" encompasses, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. In general, engineered vectors comprise an origin of replication, a multiple cloning site, and a selective marker. The vector itself is typically a nucleotide sequence, typically a DNA sequence, comprising an insert (transgene) and a larger sequence that serves as the vector's "backbone." Modern vectors may also include, in addition to the transgenic insert and backbone, additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors are called expression vectors (expression constructs) and are specifically designed to express transgenes in target cells and generally have control sequences.

[0304] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0305] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader (signal sequence) is expressed as a preprotein that participates in the secretion of the polypeptide, the DNA is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to the coding sequence. In general, "operably linked" means that the DNA sequences being linked are continuous and, in the case of a secretory leader, continuous and in reading phase. However, enhancers are not necessarily continuous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used as per conventional practice.

[0306] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. This term is mostly used for non-viral methods in eukaryotic cells. Transduction is usually used to describe the transfer of nucleic acid molecules or polynucleotides mediated by viruses. Transfection of animal cells usually involves opening temporary pores or "holes" in the cell membrane to allow the absorption of the substance. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the substance to produce liposomes, which fuse with the cell membrane and deposit their cargo therein.

[0307] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, as well as non-animal eukaryotic cells (including plant cells). Thus, transformation is the genetic alteration that occurs when bacteria or non-animal eukaryotic cells directly absorb exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane and subsequently incorporate the exogenous genetic material. Transformation can be achieved by artificial means. In order for transformation to occur, the cell or bacterium must be in a competent state, which may occur as a time-limited response to environmental conditions such as starvation and cell density.

[0308] In addition, the present invention also provides host cells transformed or transfected with the polynucleotide / nucleic acid molecules or vectors of the present technology.

[0309] As used herein, the terms "host cell" and "recipient cell" are intended to include any single cell or cell culture that can be or has been a recipient of vectors, exogenous nucleic acid molecules and polynucleotides encoding the polypeptides or binding agents of the present disclosure; and / or a recipient of the polypeptides or binding agents themselves. The introduction of the corresponding substances into the cells is carried out by transformation, transfection, etc. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Certain modifications may occur in successive generations due to natural, accidental or intentional mutations or due to environmental influences, so such progeny may not actually be completely identical (morphologically or in genomic or total DNA complement) to the parent cell, but they are still included within the scope of the terms as used herein. Suitable host cells include prokaryotic cells or eukaryotic cells, and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells such as insect cells and mammalian cells, for example, mouse, rat, macaque or human.

[0310] The polypeptides or binding agents of the present disclosure can be produced in bacteria. After expression, the polypeptide or binding agent is isolated from the E. coli cell paste as a soluble fraction and can be purified, for example, by affinity chromatography and / or size exclusion. Final purification can be performed in a manner similar to the process for purifying proteins expressed, for example, in CHO cells.

[0311] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for polypeptides or binding agents. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragili (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 402 226); 183 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0312] Host cells suitable for expressing the glycosylated polypeptides or binding agents of the present disclosure are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants and corresponding permissive insect host cells have been identified, such as from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori, which can be used as viruses according to the present disclosure, particularly for transfecting Spodoptera frugiperda cells.

[0313] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis thaliana, and tobacco can also be used as hosts. Cloning and expression vectors that can be used to produce proteins in plant cell culture are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342: 76-78; Owen et al. (1992) Bio / Technology 10: 790-794; Artsaenko et al. (1995) The Plant J 8: 745-750; and Fecker et al. (1996) Plant Mol Biol 32: 979-986.

[0314] However, of greatest interest is the vertebrate cell, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40 (e.g., ATCC CRL 1573); 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 (CV1 ATCC CRL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CRL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N. Y Acad. Sci. (1982) 383: 44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0315] In another embodiment, the present disclosure provides a method for producing a polypeptide or binding agent, the method 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.

[0316] 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 steps involved in the production of a polypeptide or binding agent of the present disclosure, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0317] When using recombinant technology, polypeptide or binding agent can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the polypeptide or binding agent is produced intracellularly, the first step is to remove particulate debris (host cells or dissolved fragments) by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a method for separating antibodies secreted into the periplasmic space of E. coli. In simple terms, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antibody is secreted into the culture medium, the supernatant from such expression system is usually first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (such as PMSF) can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0318] The polypeptides or binding agents of the present invention prepared from host cells can be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other protein purification techniques, such as ion exchange column separation, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin SEPHAROSE™ chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, can also be used, depending on the antibody to be recovered. When the polypeptides or binding agents of the present invention comprise a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) can be used for purification.

[0319] Affinity chromatography is a common purification technique. The matrix to which the affinity ligand is attached is usually agarose, but other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, can achieve faster flow rates and shorter processing times than those achieved with agarose.

[0320] The polypeptides or binding agents disclosed herein can be prepared by a variety of methods familiar to those skilled in the art, including recombinant DNA methods.

[0321] To express a polypeptide or binding agent, a nucleotide sequence encoding a polypeptide chain as 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 specific host). These elements may include regulatory sequences such as 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, and the like.

[0322] A variety of expression vectors and host cell systems known to those skilled in the art can be used to express the polypeptide chains described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage, 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 can be used. For example, a nucleotide sequence encoding any of the polypeptide chains described herein can 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 located on the same vector or a separate vector. The present disclosure is not limited by the vector or host cell used. In certain embodiments disclosed herein, the nucleic acid encoding the polypeptide chains described herein can be linked to an expression vector. If the binding agent is composed of different polypeptide chains (i.e., the first polypeptide and the second polypeptide are not identical), each such polypeptide chain can be linked to a separate vector or to the same vector. According to the present disclosure, the polypeptide chains of the binding agent can be encoded by a single vector or separate vectors (e.g., a vector set). Cells are transformed with the desired vector or vector set.

[0323] Alternatively, the polypeptide chains can be expressed separately from an in vitro transcription system or a coupled in vitro transcription / translation system or any such cell-free system.

[0324] Host cells containing the nucleotide sequence can be cultured under conditions where the corresponding RNA (mRNA, etc.) is transcribed and / or the polypeptide is expressed and secreted from the cell culture. In an exemplary embodiment, an expression vector containing a nucleotide sequence encoding a polypeptide chain described herein can be designed to contain a signal sequence that directs secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane.

[0325] Due to the inherent degeneracy of the genetic code, DNA sequences encoding identical, substantially identical, or functionally equivalent amino acid sequences can be generated and used. The nucleotide sequences of the present disclosure can be engineered using methods well known in the art to alter the nucleotide sequence for various purposes, including but not limited to cloning, processing, and / or expression of modified gene products. DNA shuffling by random fragmentation and PCR recombination of gene fragments and synthetic oligonucleotides can be used to engineer nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preferences, generate splice variants, and the like. The present disclosure encompasses codon-optimized nucleic acids encoding the polypeptide chains described herein.

[0326] In addition, the host cell strain can be selected based on its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide in a desired manner. Different host cells (e.g., CHO, HeLa, MDCK, HEK293, and W138) with specific cellular machinery and post-translational activity profiles are commercially available or available from the American Type Culture Collection (ATCC), and these cells can be selected to ensure correct modification and processing of the expressed polypeptide.

[0327] Those skilled in the art will also readily recognize that nucleic acid and peptide 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, and machines such as the ABI 431A peptide synthesizer (PE Biosystems) can be used to automatically synthesize. If desired, the amino acid sequence can be altered during the synthesis process and / or the amino acid sequence can be combined with other protein sequences to produce variant proteins.

[0328] Pharmaceutical composition

[0329] The present disclosure provides pharmaceutical compositions comprising the polypeptides disclosed herein or TGFβ superfamily ligand binding agents. Pharmaceutical compositions generally comprise the polypeptides or binding agents disclosed herein and a pharmaceutically acceptable carrier.

[0330] The preparation of pharmaceutical compositions can be carried out in a manner known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 20th edition, 2000). For example, therapeutic compounds and / or compositions are combined with one or more solid or liquid pharmaceutical carrier materials and / or additives (or auxiliary substances), and if necessary, with other pharmaceutically active compounds with therapeutic or preventive effects, to make suitable forms of administration or dosage forms, which can then be used as human or veterinary medicines. Pharmaceutical preparations can also contain additives, many of which are known in the art, such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, fragrances, thickeners, diluents, buffer substances, solvents, solubilizers, agents for realizing reservoir effects, salts for changing osmotic pressure, coating agents, or antioxidants.

[0331] The term "pharmaceutical composition" means a composition comprising a polypeptide or binding agent as described herein and at least one component comprising 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 dispersing agents, depending on the mode of administration and the nature of the dosage form.

[0332] The term "pharmaceutically acceptable carrier" is used to refer to any carrier, diluent, adjuvant, excipient or vehicle as described herein or as known in the art. The example of a suspending agent includes ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide (aluminum metahydroxide), bentonite, agar and tragacanth gum, or a mixture of these substances. It can be ensured that microbial action is prevented by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be necessary to include isotonic agents, such as sugar, sodium chloride, etc. The absorption of injectable pharmaceutical forms can be extended by using delayed absorption agents, such as aluminum monostearate and gelatin. The limiting examples of suitable carriers, diluents, solvents or vehicles include water, saline solutions, phosphate buffered saline (PBS), gelatin, oils, alcohols, polyols, their suitable mixtures, 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.

[0333] The term "pharmaceutically acceptable" means that it is suitable, within the scope of sound medical judgment, for use in contact with cells of subjects (e.g., humans and animals) without undue toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit / risk ratio.

[0334] As used herein, "pharmaceutically acceptable carrier" or "drug carrier" is known in the art and includes, but is not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / 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 solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements, such as those based on Ringer's dextrose, etc. Preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, etc., may also be present.

[0335] Pharmaceutically acceptable carriers may include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. 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 and oral administration. In other embodiments, the carrier is suitable for topical administration or administration via inhalation. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersants and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersants. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is contemplated that they will be used in the pharmaceutical compositions provided herein. Supplementary active compounds may also be incorporated into the composition. For example, the pharmaceutical compositions provided herein may also include at least one additional therapeutic agent, as discussed further below.

[0336] The pharmaceutical compositions described herein can be administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0337] In some embodiments, the pharmaceutical compositions provided herein can be administered orally, for example, in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example, in the form of suppositories.

[0338] In other embodiments, the pharmaceutical compositions provided herein can be administered parenterally, for example, subcutaneously, intramuscularly, or intravenously, in the form of injection or infusion solutions. Other suitable administration forms are, for example, transdermal or topical administration, for example, in the form of ointments, creams, tinctures, sprays, or transdermal therapeutic systems; or administration by inhalation, in the form of nasal sprays or aerosol mixtures; or, for example, microcapsules, implants, or wafers.

[0339] Pharmaceutical compositions must generally be sterile and stable under conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for 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, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, suitable fluidity can be maintained. In many cases, it is preferred to include an isotonic agent in the composition, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride. Extended absorption of injectable compositions can be achieved by including an agent that delays absorption (e.g., monostearate and gelatin) in the composition. In addition, compounds can be administered in delayed release formulations, for example, in compositions comprising sustained-release polymers. Compounds can be prepared with carriers that prevent rapid release, such as controlled-release formulations comprising 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-polyglycolic acid copolymers (PLG).

[0340] Many methods for preparing such formulations are generally known to those skilled in the art. A desired amount of active compound (such as polypeptide or binding agent provided herein) and a composition listed above or their combination can be mixed in an appropriate solvent, as needed, followed by filtration sterilization to prepare a sterile injection solution. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle containing an alkaline dispersion medium and other required components listed above. In the case of sterile powders for the preparation of sterile injection solutions, common preparation methods are vacuum drying and freeze drying, which obtain powders of active ingredients and any other required components from their previous sterile filtered solutions. The compound can also be formulated with one or more other compounds that enhance its solubility.

[0341] In some embodiments, the pharmaceutical compositions of the present disclosure comprise sterile injectable solutions. In some embodiments, sterile injectable solutions comprise sterile powders for reconstitution with an acceptable solution (e.g., water).

[0342] It is often advantageous to formulate compositions (such as parenteral compositions) in dosage unit form for ease of administration and uniformity of dosage. The term "unit dosage form" refers to physically discrete units suitable as single doses for human subjects and other animals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier. The specifications of the dosage unit forms of the present invention can vary and are dictated by and directly dependent on: (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the art of compounding such therapeutic compounds for the prevention or treatment of diseases or disorders associated with the TGFβ superfamily. Dosage will be discussed further below.

[0343] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

[0344] For any compound, the therapeutically effective dose can be initially estimated in a cell culture assay or in an animal model (such as a mouse, rat, rabbit, dog or pig). An animal model can also be used to determine the administration concentration range and route. Such information can then be used to determine the useful dose and route 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 the active ingredient that improves symptoms or symptoms. Therapeutic efficacy and toxicity can be determined by standard drug procedures in cell cultures or experimental animals, such as by calculating and comparing ED50 (dose effective for 50% of the population treatment) and LD50 (dose lethal to 50% of the population). Any pharmaceutical composition described herein can be applied to any subject in need of treatment, including but not limited to mammals, such as dogs, cats, cattle, horses, rabbits, monkeys, and especially people.

[0345] The terms "effective dose," "effective dosage," and "effective amount" are used interchangeably and 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 or ameliorate or at least partially prevent the 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 typically results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of symptom-free periods of the disease, or prevention of damage or disability resulting from the disease. In some embodiments, a therapeutically effective amount is an amount or dosage of a polypeptide or binding agent or composition that prevents or treats a TGFβ superfamily-related disease or condition in a subject, as described herein. In some embodiments, an effective amount is an amount or dosage 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.

[0346] The amount or dosage effective for this use will depend on the condition to be treated (indication), the polypeptide or binding agent being delivered, the therapeutic 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 for administration to the patient in one or more doses to obtain the optimal therapeutic effect.

[0347] Depending on the factors mentioned above, typical dosages can range from about 0.1 μg / kg up to about 30 mg / kg or higher. In specific embodiments, dosages can range from 1.0 μg / kg to about 20 mg / kg, optionally from 10 μg / kg to about 10 mg / kg or from 100 μg / kg to about 5 mg / kg. Dosages are discussed further below.

[0348] How to use

[0349] The formulations described herein can be used as pharmaceutical compositions for treating, ameliorating, and / or preventing a disease / condition as described herein in a subject in need thereof. The term "treat" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes applying or administering a formulation to the body, isolated tissue, or cells of a patient suffering from a disease / condition, symptoms of a disease / condition, or a predisposition to a disease / condition, with the purpose of curing, rehabilitating, alleviating, mitigating, altering, remedying, improving, modifying, or influencing the disease, symptoms of a disease, or a predisposition to a disease.

[0350] Any reference herein to methods of treatment may also be construed as a reference to the use of an ActRIIB-ECD polypeptide described herein, or a TGFβ superfamily ligand-binding agent comprising the same, in such methods of treatment.

[0351] As used herein, the term "ameliorate" refers to any improvement in the disease state of a patient suffering from a disease / disorder specified below by administering a polypeptide or binding agent or pharmaceutical composition according to the present disclosure to a subject. For example, the term "ameliorate" means to reduce, inhibit, attenuate, weaken, prevent or stabilize the development or progression of a disease. Such improvement can also be seen as a slowing down or stopping of the progression of a patient's disease / disorder. As used herein, the term "prevent" means to avoid the occurrence or recurrence of a patient suffering from a disease / disorder specified below by administering a polypeptide or binding agent or pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0352] 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 / condition in question. In some embodiments, the polypeptides or binding agents of the present disclosure and compositions thereof may be used to prevent, treat, or ameliorate a TGFβ superfamily-associated disease or condition. Thus, methods are provided for preventing or treating a TGFβ superfamily-associated disease or condition in a subject, comprising administering a therapeutically effective amount of a polypeptide or binding agent or pharmaceutical composition described herein. The polypeptides and binding agents are typically administered in the form of a pharmaceutical composition. A subject may be in need of such treatment, i.e., has, is suspected of having, or is 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.

[0353] As used herein, the terms "inhibition" and "inhibiting" generally refer to reducing, slowing, limiting, delaying, inhibiting, blocking, neutralizing, hindering, or preventing a process, such as, but not limited to, reducing or slowing the progression, growth, or spread of a disease or disorder.

[0354] In some embodiments of the present disclosure, "treating" refers to neutralizing the biological activity of excess activin A, activin B, GDF-8, and / or GDF-11. This can be determined by: appropriate clinical improvement variables; 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 substantial inhibition of BMP-9 and / or BMP-10); or other measures appropriate to the disease or condition being treated.

[0355] In some embodiments, improvement is determined by comparing clinical variables to values ​​measured before treatment, or alternatively, to typical values ​​measured in healthy adults. In certain embodiments, treatment or prevention is within the scope 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 a placebo group member, historical control, or between subsequent trials conducted on the same subject.

[0356] The term "subject" includes living organisms suffering from a TGFβ superfamily-related disease or disorder, or living organisms susceptible to or at risk thereof. 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, such as mammals, such as primates, such as humans, that are susceptible to a state characterized by a TGFβ superfamily-related disease or disorder (such as pulmonary hypertension, fibrosis, muscle weakness and atrophy, metabolic disorders and / or cardiometabolic diseases, bone damage, or low red blood cell levels). Animals can also be animal models of the disease, such as mouse models, xenotransplant recipients, etc. In certain embodiments, the subject is a human.

[0357] There are no particular limitations on the dosage of the polypeptide or binding agent used in the compositions and methods of the present disclosure. Exemplary dosages include milligrams or micrograms of polypeptide or binding agent per kilogram of subject or sample weight (e.g., about 50 micrograms per kilogram to about 500 milligrams per kilogram, about 1 milligram per kilogram to about 100 milligrams per kilogram, about 1 milligram per kilogram to about 50 milligrams per kilogram, about 1 milligram per kilogram to about 10 milligrams per kilogram, or about 3 milligrams per kilogram to about 5 milligrams per kilogram). Additional exemplary dosages 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, for example, once a day or twice a day, or lower or higher amounts.

[0358] In some embodiments, the dosage range for adults is generally 0.005 mg to 10 g / day. The polypeptides or binding agents and compositions thereof can be provided in unit dosage form, for example, in units effective at such dosages or multiples thereof, for example, units containing 5 mg to 500 mg, typically about 10 mg to 200 mg. Dosage units can include, 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, 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). mg) of a polypeptide or binding agent or composition described herein.

[0359] It should be understood that the effective amount of a polypeptide or binding agent for treating a disease or illness varies according to the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian determines the appropriate amount and dosage regimen. It should be understood that the dosage or amount of the polypeptide or binding agent used alone or in combination with one or more active compounds to be administered depends on the individual case, and should be adapted to the individual case as a rule to achieve the best effect. Administration and administration regimens are within the scope of those skilled in the art, and appropriate dosages depend on a variety of factors within the knowledge of the general technical physician, veterinarian, or researcher (e.g., see Wells et al., eds., Pharmacotherapy Handbook, 2nd ed., Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000)). For example, the dosing and administration regimen will depend on the nature and severity of the disease or condition being treated, as well as on the sex, age, weight and individual responsiveness of the human or animal being treated, on the efficacy and duration of action of the compound being used, on whether the treatment is acute, chronic or prophylactic, and / or on whether other active compounds are being administered in addition to the therapeutic molecule.

[0360] Known procedures can be used to administer polypeptides or binding agents or compositions provided herein in dosages and time periods that effectively achieve the desired purpose. Dosage regimens can be adjusted to provide optimal therapeutic responses. For example, several separate dosages can be administered daily, weekly, biweekly, or monthly, or the dosage can be proportionally reduced as indicated by the urgency of the treatment situation. In some embodiments, polypeptides or binding agents or compositions are administered in a therapeutically effective dose sufficient to prevent or treat a TGFβ superfamily-related disease or illness (e.g., pulmonary hypertension, fibrosis, muscle weakness and atrophy, metabolic disorders and / or cardiometabolic diseases, bone damage, and / or low red blood cell levels) of a subject.

[0361] In some embodiments, according to the methods of the present disclosure, one or more symptoms of development or progression of a TGFβ superfamily-related disease or disorder in a subject is reduced 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%.

[0362] 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 therapy or therapeutic agent may be administered before, after, or simultaneously with the administration of the polypeptide or binding agent or composition described herein. In some embodiments, the additional therapy or therapeutic agent is formulated in the same composition as the polypeptide or binding agent. In other embodiments, the additional therapy or therapeutic agent is administered alone. 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, such as an antibody, antibody fragment, antigen binding fragment, soluble TGFβ superfamily ligand capture agent, etc.; another agent that binds to or inhibits one or more additional targets, etc.

[0363] Alternatively, in some embodiments, the polypeptide or binding agent can be conjugated to a detectable or diagnostic moiety that can be used to track the polypeptide or binding agent or cells or tissues expressing a TGFβ superfamily ligand and / or another target. In some such embodiments, a method of diagnosing a TGFβ superfamily-associated disease or condition is provided, 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, thereby diagnosing a disease or condition associated with TGFβ superfamily signaling (e.g., overexpression of activin A, activin B, GDF-8, or GDF-11).

[0364] TGFβ superfamily-related diseases and disorders

[0365] The polypeptides or binding agents described herein and their pharmaceutical compositions can be used to prevent or treat diseases and disorders associated with the TGFβ superfamily. Thus, methods are provided for preventing or treating a disease or disorder associated with TGFβ superfamily ligand signaling in a subject, comprising administering a therapeutically effective amount of a polypeptide, binding agent, or pharmaceutical composition described herein. The polypeptides and binding agents are typically administered in the form of a pharmaceutical composition. A subject may be in need of such treatment, i.e., has, is suspected of having, or is at risk of developing a disease or disorder associated with TGFβ superfamily signaling, as described herein.

[0366] As used herein, the term "TGFβ superfamily ligand signaling-related disease or disorder" refers to diseases and disorders that can be improved by inhibiting one or more TGFβ superfamily ligands (particularly activin A, activin B, GDF-8 and / or GDF-11 activity). TGFβ superfamily-related diseases or disorders include, but are not limited to, diseases and disorders associated with overexpression or overactivation of TGFβ superfamily ligands (particularly activin A, activin B, GDF-8 and / or GDF-11). In some embodiments, TGFβ superfamily-related diseases or disorders are mediated by activin A and / or activin B. In one embodiment, the disease or disorder to be treated is mediated by activin A. In one embodiment, the disease or disorder to be treated is mediated by activin B. In one embodiment, the disease or disorder to be treated is mediated by GDF-8. In one embodiment, the disease or disorder to be treated is mediated by GDF-11. In another embodiment, the disease or condition to be treated is mediated by a combination of activin A, activin B, GDF-8, and GDF-11.

[0367] Examples of TGFβ superfamily-associated diseases or conditions that can be prevented or treated according to the present disclosure include, but are not limited to, pulmonary hypertension, fibrosis, muscle weakness and atrophy, metabolic disorders and / or cardiometabolic diseases, bone damage and / or low red blood cell levels, and vascular diseases and vasculopathy.

[0368] Metabolic disorders and cardiometabolic diseases

[0369] In some embodiments, the polypeptides or binding agents described herein are used to treat or prevent metabolic disorders and / or cardiometabolic diseases. Non-limiting examples of metabolic disorders include diabetes (including type 1 diabetes and type 2 diabetes), prediabetes, diabetic complications (such as, for example, retinopathy, nephropathy or neuropathy, diabetic foot, ulcers, macroangiopathy), metabolic acidosis or ketosis, hyperglycemia, reactive hypoglycemia, hyperinsulinemia, glucose metabolism disorders, insulin resistance, metabolic syndrome, dyslipidemia, atherosclerosis and related diseases, obesity, prediabetes, hypertension, chronic heart failure, acute renal failure, edema and hyperuricemia. Non-limiting examples of cardiometabolic diseases include heart attack, stroke, diabetes, prediabetes, insulin resistance, non-alcoholic fatty liver disease, chronic renal failure, heart failure and / or low ejection fraction, including heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). In some embodiments, the cardiometabolic disease is HFrEF. In some embodiments, the cardiometabolic disease is HFpEF.

[0370] Treatment of obesity or overweight conditions and their associated comorbidities (such as type 2 diabetes, dyslipidemia, hyperglycemia, cardiovascular disease, and other metabolic disorders) represents a significant unmet medical need. Obesity is a risk factor for overall mortality and is estimated to have caused 3.4 million deaths worldwide in 2010 (Lim et al. 2012). Obesity, diabetes, hyperglycemia, and related conditions are high risk factors for serious or life-threatening complications (such as heart disease, stroke, kidney problems, nerve damage, and eye conditions). In particular, cardiometabolic diseases such as stroke, heart attack, and cardiovascular disease can be improved by lowering blood glucose levels or reducing weight in patients with type 2 diabetes or obesity. Pharmacological agents that can improve body composition by altering the ratio of lean body mass to fat mass, thereby achieving weight loss or reducing central fat accumulation, and improving the patient's glycemic status are urgently sought. There is also a need for therapies that address heart failure associated with left ventricular dysfunction (such as heart failure with preserved ejection fraction (HFpEF)). HFpEF accounts for more than half of heart failure cases worldwide, and there is an urgent need for therapies to treat HFpEF and its associated risk factors, such as obesity.

[0371] There is a need for pharmacological agents that can improve body composition by altering the ratio of lean to fat mass, thereby achieving weight loss or reducing central fat accumulation and improving glycemic status in patients. Increased muscle mass has been reported in myostatin knockout mice and in wild-type mice in which myostatin is sequestered by soluble ActRIIB, and this increase has been associated with improved whole-body insulin sensitivity and resistance to diet-induced genetic obesity (Guo et al., 2009, Akpan et al., 2009). Inhibition of ActRIIB with antibodies has been shown to reduce white adipose tissue in mice fed a normal or high-fat diet, while increasing skeletal muscle mass (Fournier et al., 2012).

[0372] In some embodiments, the present disclosure provides methods for treating a subject with a metabolic disorder, comprising administering to a subject in need thereof an effective amount of an ActRIIB-ECD polypeptide as described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide. For example, methods for treating type 2 diabetes, metabolic syndrome, hyperglycemia, and obesity are provided. Thus, in some embodiments, methods for preventing or treating metabolic disorders or conditions such as, but not limited to, type 1 diabetes, type 2 diabetes, diabetic complications (such as, for example, retinopathy, nephropathy, or neuropathy, diabetic foot, ulcers, macroangiopathy), metabolic acidosis or ketosis, reactive hypoglycemia, hyperinsulinemia, glucose metabolism disorders, insulin resistance, metabolic syndrome, dyslipidemia, atherosclerosis and related diseases, obesity, prediabetes, hypertension, chronic heart failure, acute renal failure, edema, or hyperuricemia are provided.

[0373] In some embodiments, the present disclosure provides methods of increasing lean body mass in a subject having a metabolic disorder, the methods comprising administering to a subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0374] In some embodiments, the present disclosure provides methods of increasing muscle mass in a subject having a metabolic disorder, the methods comprising administering to a subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0375] In some embodiments, the present disclosure provides methods of reducing white fat and / or increasing brown fat in a subject in need thereof (e.g., a subject with a metabolic disorder), the methods comprising administering to the subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0376] In some embodiments, the present disclosure provides methods of reducing total body fat mass (FM) in a subject in need thereof (e.g., a subject with a metabolic disorder), the method comprising administering to the subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0377] In some embodiments, the present disclosure provides methods of reducing total body fat mass (FM) while increasing muscle mass in a subject in need thereof (e.g., a subject with a metabolic disorder such as obesity), the method comprising administering to the subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0378] In some embodiments, the present disclosure provides methods of improving insulin sensitivity in a subject in need thereof (e.g., a subject with a metabolic disorder), comprising administering to a subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0379] In some embodiments, the present disclosure provides methods of increasing energy expenditure in a subject in need thereof (e.g., a subject with a metabolic disorder), comprising administering to the subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein, or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0380] In some embodiments, the present disclosure provides a method for treating a subject with a cardiometabolic disease, the method comprising administering to a subject in need thereof an effective amount of an ActRIIB-ECD polypeptide as described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide. Non-limiting examples of cardiometabolic diseases include heart attack, stroke, diabetes, insulin resistance, non-alcoholic fatty liver disease, and chronic renal failure. In some such embodiments, the present disclosure provides a method for reducing the risk of cardiovascular death in subjects with type 2 diabetes and established cardiovascular disease. In some such embodiments, the present disclosure provides a method for reducing the risk of death and / or hospitalization in patients with heart failure and / or low ejection fraction. In some such embodiments, the present disclosure provides a method for reducing the risk of death and / or hospitalization in people with heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF). In some such embodiments, the present disclosure provides a method for reducing the risk of cardiovascular death and / or hospitalization in adults. In some such embodiments, the present disclosure provides a method for reducing blood glucose levels in subjects with type 2 diabetes. In some such embodiments, the present disclosure provides methods of treating or preventing metabolic disorders (such as diabetes) and / or improving glycemic control in subjects with moderate renal impairment or stage 3 chronic kidney disease.

[0381] In some embodiments, the disclosure provides methods of improving body composition, e.g., for the treatment of central adiposity, obesity or overweight conditions and related comorbidities, comprising administering to a subject in need thereof an effective amount of an ActRIIB-ECD polypeptide described herein or a TGFβ superfamily ligand binding agent comprising the ActRIIB-ECD polypeptide.

[0382] In some embodiments, the disclosure provides methods of treating type II diabetes, e.g., by improving glycemic control or increasing insulin sensitivity, comprising administering to a subject in need...

Claims

1. A polypeptide comprising an amino acid sequence at least 85% identical to an activin type IIB receptor (ActRIIB) extracellular domain (ECD) variant, wherein the ActRIIB ECD variant comprises an amino acid substitution at a position corresponding to position 33 of SEQ ID NO:

2.

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

3. The polypeptide of claim 1 or claim 2, wherein the amino acid substitution is selected from the group consisting of 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 polypeptide of claim 4, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 6, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 8, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 10, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 12, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 14, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 16, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 18, wherein the ActRIIB ECD variant (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) comprises or consists 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 polypeptide of claim 20, wherein the ActRIIB ECD variant (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) comprises or consists of the amino acid sequence of SEQ ID NO:

18.

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

2.

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

2.

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

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

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

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

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

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

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 murine Fc domain monomer.

32. The polypeptide of any one of claims 27 to 31, wherein the Fc domain monomer is engineered to reduce aggregation of dimers of the polypeptide or to modulate the stability of dimers of the polypeptide.

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

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

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 (DL) at position 358.

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

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

38. The polypeptide of any one of claims 27 to 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. 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. The polypeptide of any one of claims 30 to 39, wherein the Fc domain monomer is of the IgG1 isotype.

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 dimers.

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

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

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

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

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

49. 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. The polypeptide of any one of claims 1 to 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. 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: 4-62.

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 NOs: 10-18, or a 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 thereto.

53. 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 thereto.

54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide 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 an amino acid sequence selected from SEQ ID NOs: 174-251.

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

56. The polypeptide of any one of claims 1 to 54, wherein the polypeptide 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 an amino acid sequence selected from SEQ ID NOs: 175, 176, 180, 204-214 and 230-234.

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

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

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

60. The polypeptide of any one of claims 1 to 54, wherein the 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 thereto.

61. The polypeptide of any one of claims 1 to 54, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

231.

62. The polypeptide of any one of claims 1 to 54, wherein the 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 thereto.

63. The polypeptide of any one of claims 1 to 54, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

234.

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 the 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 located at the N-terminus of the ActRIIB-ECD.

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

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. The polypeptide of claim 67, wherein the targeting agent, the therapeutic moiety, the detectable moiety, or the 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 drug, a nanoparticle-based carrier, a polymer-conjugated drug, a nanocarrier, an imaging agent, a stabilizer, a drug, a nanocarrier, or a dendrimer.

69. The polypeptide of any one of claims 1 to 68, wherein the polypeptide forms a dimer comprising a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide being 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. A TGFβ superfamily ligand binding agent comprising a first polypeptide as described in any one of claims 1 to 69 and a second polypeptide as described in any one of claims 1 to 69, 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.

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 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 thereto.

72. 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 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 thereto.

73. 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 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 thereto.

74. 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 at least 95%, 96%, 97%, 98% or 99% identical thereto.

75. 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 at least 95%, 96%, 97%, 98% or 99% identical thereto.

76. 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 at least 95%, 96%, 97%, 98% or 99% identical thereto.

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

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

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

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

81. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the polypeptide does not substantially inhibit human BMP-9 and / or BMP-10 signaling.

82. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the polypeptide has an inhibitory potency on human BMP-9 and / or BMP-10 signaling 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 on human BMP-9 and / or BMP-10 signaling.

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

84. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the polypeptide has an inhibitory potency on human BMP-9 and / or BMP-10 signaling that 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.

85. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the polypeptide has an inhibitory potency on human BMP-9 and / or BMP-10 signaling that 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. The polypeptide of any one of claims 1 to 69 or the binding agent of any one of claims 70 to 76, wherein the polypeptide has the same or substantially the same inhibitory potency against one or more of human activin-A, activin-B, GDF-8, and GDF-11 as human wild-type ActRIIB-ECD against the same corresponding ligand.

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

88. 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 corresponding ligands; and / or wherein the relative inhibitory potency against BMP-9 and / or BMP-10 is decreased compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same ligands.

89. 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 a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same corresponding ligands; and / or wherein the relative inhibitory potency against BMP-9 and / or BMP-10 is decreased compared to the inhibitory potency of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same ligands.

90. 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 the human wild-type ActRIIB-ECD or the polypeptide having the amino acid sequence set forth in SEQ ID NO: 171 against the same corresponding ligand.

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

171.

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

171.

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

94. nucleic acid molecules as claimed in claim 93, it also comprises the sequence of setting forth in the SEQ ID NO:297 that is positioned at the 5 ' end of described nucleic acid molecules.

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

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

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

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

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

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

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

102. The pharmaceutical composition of any one of claims 98 to 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 the subject.

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

104. 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 as described in any one of claims 1 to 69, a binding agent as described in any one of claims 70 to 92, or a pharmaceutical composition as described in any one of claims 98 to 102.

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

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 for 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 as described in any one of claims 1 to 69, a binding agent as described in any one of claims 70 to 92, or a pharmaceutical composition as described in any one of claims 98 to 102.

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

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 disease, bone damage, and low red blood cell levels.

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

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

112. The method of claim 109, wherein the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, liver fibrosis, lung fibrosis, kidney fibrosis, myelofibrosis, systemic sclerosis, skin fibrosis, cardiac fibrosis, myeloid fibrosis, corneal fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthritis, arthrofibrosis, tissue fibrosis, a fibroproliferative disorder, or a connective tissue disorder.

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

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

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

116. 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, osteosclerosis, bone fracture, bone cancer or metastasis-related bone loss, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low gravity-related bone loss, or immobilization-related bone loss.

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

119. 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 as described in any one of claims 1 to 69, a binding agent as described in any one of claims 70 to 92, or a pharmaceutical composition as described in any one of claims 98 to 102.

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

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

122. The method of any one of claims 104 to 121, wherein 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 hematological complications in the subject.

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