Activin receptor type IIB variants and uses thereof

Abstract

Polypeptides are provided that include activin receptor type IIB (ActRIIB) ectodomain (ECD) variants. In some embodiments, the polypeptides of the present disclosure include ActRIIB-ECD variants fused to Fc domain portions. The present disclosure also provides pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions associated with TGFβ superfamily ligand signaling, such as pulmonary hypertension, fibrosis, muscle weakness and atrophy, metabolic and / or cardiometabolic diseases, bone damage, and / or low red blood cell levels (such as anemia).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international PCT application claiming benefit of priority from U.S. Provisional Application No. 63 / 304,478, filed January 28, 2022, U.S. Provisional Application No. 63 / 397,773, filed August 12, 2022, U.S. Provisional Application No. 63 / 416,852, filed October 17, 2022, and U.S. Provisional Application No. 63 / 420,999, filed October 31, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (35PH_002_04WO_SeqList_ST26.xml, size: 417,164 bytes, and created on January 27, 2023) are incorporated herein by reference in their entirety.

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

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

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

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

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

[0008] In some embodiments, the present disclosure provides a polypeptide comprising: (a) an activin receptor type IIB (ActRIIB) ectodomain (ECD) variant, (b) a peptide linker comprising at least 10 amino acids, and (c) an Fc domain monomer. In some embodiments, the ActRIIB ECD comprises one or more amino acid substitutions at positions selected from G27, Q29, D30, K31, S38, D57, F58, V75, and F77 compared to human wild-type ActRIIB-ECD of SEQ ID NO: 2.

[0009] In some embodiments, the ActRIIB ECD comprises the amino acid substitution G27D. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20. In some embodiments, the ActRIIB ECD comprises the amino acid substitution Q29Y. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14. In some embodiments, the ActRIIB ECD comprises the amino acid substitution D30Q. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15. In some embodiments, the ActRIIB ECD comprises the amino acid substitution K31Y. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16. In some embodiments, the ActRIIB ECD comprises the amino acid substitution S38R. In some embodiments, the ActRIIB ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the ActRIIB ECD comprises the amino acid substitution D57E. In some embodiments, the ActRIIB ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

[0010] In some embodiments, the ActRIIB ECD comprises an amino acid substitution at position F58 selected from F58D, F58E, F58Y, F58K, F58Q, F58N, F58R, F58H, and F58W. In some embodiments, the ActRIIB ECD comprises the amino acid substitution F58D. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6. In some embodiments, the ActRIIB ECD variant comprises the amino acid substitution F58E. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5. In some embodiments, the variant comprises the amino acid substitution F58Y. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7. In some embodiments, the ActRIIB ECD comprises the amino acid substitution F58K. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8. In some embodiments, the variant comprises the amino acid substitution F58Q. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9. In some embodiments, the variant comprises the amino acid substitution F58W. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10. In some embodiments, the variant comprises the amino acid substitution F58N. In some embodiments, the ActRIIB ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11. In some embodiments, the variant comprises the amino acid substitution F58H. In some embodiments, the ActRIIB ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the variant comprises the amino acid substitution F58R.In some embodiments, the ActRIIB ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0011] In some embodiments, the ActRIIB ECD comprises the amino acid substitution V75Q. In some embodiments, the ActRIIB ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. In some embodiments, the ActRIIB ECD comprises the amino acid substitution F77D. In some embodiments, the ActRIIB ECD comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19.

[0012] In some embodiments, the ActRIIB-ECD variant further comprises one or more additional amino acid substitutions. In some embodiments, the ActRIIB-ECD variant further comprises the following amino acid at the N-terminus: GRGEA (SEQ ID NO: 23). In some embodiments, the ActRIIB-ECD variant further comprises a 3-amino acid stretch of alanine-proline-threonine (APT) at the C-terminus.

[0013] In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, the following structure: ActRIIB-ECD-peptide linker-Fc domain monomer.

[0014] In some embodiments, the Fc domain monomer is an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc domain monomer is a human Fc domain monomer or a mouse Fc domain monomer. In some embodiments, the Fc domain monomer is engineered to reduce aggregation or modulate the stability of a polypeptide dimer. 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.

[0015] 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: 134-173 and 338. In some embodiments, the Fc domain monomer comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 134-173 and 338. In some embodiments, the Fc domain monomer is (a) an IgG1 isotype and comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135 or SEQ ID NO: 134, or (b) an IgG2 isotype and comprises or consists of the amino acid sequence set forth in SEQ ID NO: 157. In some embodiments, the Fc domain monomer forms a dimer.

[0016] In some embodiments, the peptide linker is glycine-rich. In some embodiments, the peptide linker is 10 to 40 amino acids in length. In some embodiments, the linker is at least 10 amino acids in length, at least 14 amino acids in length, at least 19 amino acids in length, or at least 39 amino acids in length. In some embodiments, the linker is 10 amino acids in length, 14 amino acids in length, 19 amino acids in length, or 39 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 34, 54, 59, or 63.

[0017] In some embodiments, ActRIIB-ECD comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 4-22, 331, 332, and 24-33. In some embodiments, ActRIIB-ECD comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 4-22, 331, 332, and 24-33. In some embodiments, ActRIIB-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, ActRIIB-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 8, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, ActRIIB-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 9, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0018] 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: 174-254, 333, and 339-341. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 174-254, 333, and 339-341. 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: 186, 190-194, 232-233, and 247-248. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 186, 190-194, 232-233, and 247-248. In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 186, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polypeptide comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 213-216 and 242-244. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 213-216 and 242-244. In some embodiments, the polypeptide comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 220-223, 253, and 254. In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOs: 220-223, 253, and 254.

[0019] In some embodiments, the polypeptide further comprises an albumin-binding domain, a fibronectin domain, or a human serum albumin domain fused to the N-terminus or C-terminus of ActRIIB-ECD via a linker. In some embodiments, the polypeptide further comprises a signal peptide of SEQ ID NO: 1 at the N-terminus of ActRIIB-ECD. In some embodiments, the polypeptide is conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, or a diagnostic moiety.

[0020] In some embodiments, the targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety comprises an antibody or antigen-binding fragment thereof, a binding agent with affinity for another member of the TGFβ superfamily or for another therapeutic target, a radiotherapeutic agent, an imaging agent, a fluorescent moiety, a cytotoxic agent, an antimitotic agent, a nanoparticle-based carrier, a polymer-conjugated drug, a nanocarrier, an imaging agent, a stabilizing agent, a drug, a nanocarrier, or a dendrimer.

[0021] In some embodiments, the polypeptides form a dimer comprising a first polypeptide and a second polypeptide linked by at least one disulfide bond between an Fc domain monomer of the first polypeptide and an Fc domain monomer of the second polypeptide.

[0022] 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. In some embodiments, the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 174-254, 333, and 339-341, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0023] In some embodiments, the first polypeptide and the second polypeptide comprise or consist of an amino acid sequence selected from SEQ ID NOs: 186, 190-194, 232-233, and 247-248, 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: 186, 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 an amino acid sequence selected from SEQ ID NOs: 213-216, and 242-244, 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 an amino acid sequence selected from SEQ ID NOs: 220-223, 253, and 254, or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0024] In some embodiments, the polypeptide or binding agent binds to human activin A, activin B, GDF-8, GDF-11, and / or bone morphogenetic protein (BMP)-10 and exhibits reduced binding to human BMP-9 compared to the binding of human wild-type ActRIIB-ECD to human BMP-9. In some embodiments, the polypeptide or binding agent does not substantially bind to human BMP-9. In some embodiments, the polypeptide or binding agent inhibits signaling of one or more of human activin A, activin B, GDF-8, GDF-11, and BMP-10. In some embodiments, the polypeptide or binding agent does not inhibit human BMP-9 signaling.

[0025] In some embodiments, the inhibitory potency of the polypeptide or binding agent against human BMP-9 signaling is about 100-fold, about 200-fold, or about 300-fold lower than the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 signaling. In some embodiments, the inhibitory potency of the polypeptide or binding agent against one or more of human activin A, activin B, GDF-8, GDF-11, and BMP-10 is substantially the same as or increased compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same respective ligand(s). In some embodiments, the inhibitory potency of the polypeptide against one or more of human activin A, activin B, GDF-8, GDF-11, and BMP-10 is increased by about 2-fold, about 3-fold, about 4-fold, or about 5-fold or more compared to the inhibitory potency of human wild-type ActRIIB-ECD against the same respective ligand(s).

[0026] In some embodiments, the disclosure provides nucleic acid molecules encoding the polypeptides described herein. In some embodiments, the nucleic acid sequence 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: 256-330, 334, or 342-344. In some embodiments, the nucleic acid sequence comprises or consists of the nucleic acid sequence of any one of SEQ ID NOs: 256-330, 334, or 342-344. In some embodiments, the nucleic acid further comprises the sequence set forth in SEQ ID NO: 255 at the 5' end of the nucleic acid molecule.

[0027] In some embodiments, the present disclosure provides a vector comprising a nucleic acid described herein. In some embodiments, the present disclosure provides a host cell comprising a nucleic acid molecule or vector described herein, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0028] In some embodiments, the present disclosure provides methods for preparing a polypeptide described herein, comprising: (a) providing a host cell comprising a nucleic acid molecule or vector described herein; (b) culturing the host cell under conditions that allow expression of the polypeptide; and (c) recovering the expressed polypeptide from the culture.

[0029] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a polypeptide or binding agent described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition is formulated for administration by injection or infusion. In some embodiments, the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

[0030] In some embodiments, the polypeptide or binding agent does not cause a vascular complication in the subject and / or does not increase vascular permeability or leakage in the subject, hi some embodiments, the polypeptide or binding agent does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and / or does not cause a hematologic complication in the subject.

[0031] In some embodiments, the present disclosure provides kits comprising a polypeptide or binding agent described herein, or a pharmaceutical composition described herein, and, optionally, instructions for use.

[0032] In some embodiments, the present disclosure provides a method of treating or preventing a disease or condition associated with TGFβ superfamily ligand signaling in a subject in need thereof, the method comprising administering to the subject a polypeptide, binding agent, or pharmaceutical composition described herein. In some embodiments, the subject is a human. In some embodiments, the TGFβ superfamily ligand is one or more of activin A, activin B, GDF-8, GDF-11, and BMP-10.

[0033] In some embodiments, the present disclosure provides methods of treating or preventing a disease or condition associated with or mediated by activin A, activin B, GDF-8, GDF-11, and / or BMP-10 in a subject, the method comprising administering to the subject a polypeptide, binding agent, or pharmaceutical composition described herein. In some embodiments, the disease or condition is characterized by overexpression or overactivation of activin A and / or activin B and / or GDF-8 and / or GDF-11.

[0034] In some embodiments, the disease or condition is selected from pulmonary hypertension (PH), fibrosis, muscle weakness or atrophy, metabolic disorders, cardiometabolic disease, bone damage, and low red blood cell levels.

[0035] In some embodiments, the PH is pulmonary arterial hypertension (PAH). In some embodiments, the PAH is idiopathic PAH, hereditary PAH, or PAH associated with infection, congenital heart abnormality, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, chronic obstructive pulmonary disease, autoimmune disorders (e.g., scleroderma or lupus), or drug use (e.g., cocaine or methamphetamine use).

[0036] In some embodiments, the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, kidney fibrosis, myelofibrosis, systemic sclerosis, dermal fibrosis, cardiac fibrosis, myelofibrosis, corneal fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthrofibrosis, arthrofibrosis, tissue fibrosis, a fibromatous proliferative disorder, or a connective tissue disorder.

[0037] 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.

[0038] In some embodiments, the metabolic disorder is obesity, type 1 diabetes, type 2 diabetes, or prediabetes.

[0039] In some embodiments, the cardiometabolic disease or condition is heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

[0040] In some embodiments, bone damage includes bone demineralization, osteoporosis (e.g., primary or secondary), osteopenia, osteopetrosis, fracture, bone loss associated with bone cancer or cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with treatment for obesity, low gravity-related bone loss, or immobility-related bone loss.

[0041] In some embodiments, the disease or condition of low blood cell levels is anemia or blood loss.

[0042] In some embodiments, the present disclosure provides a method of reducing or inhibiting activin A, activin B, GDF-8, GDF-11, and / or BMP10 signaling in a subject in need thereof, without substantially reducing or inhibiting BMP9 signaling in the subject, the method comprising administering to the subject a polypeptide, binding agent, or pharmaceutical composition described herein. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0043] In some embodiments, the method does not cause vascular complications in the subject, does not increase vascular permeability or leakage in the subject, does not increase red blood cell mass, does not increase hemoglobin, does not cause thrombocytopenia, and / or does not cause hematologic complications in the subject.

[0044] Further scope, applicability, and advantages of the present technology will become apparent from the non-limiting detailed description given below. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the present technology, is given by way of example only, with reference to the accompanying drawings.

[0045] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0046] For a better understanding of the technology, and to show more clearly how it may be put into practice, reference is now made, by way of example, to the accompanying drawings which illustrate aspects and features according to non-limiting embodiments of the technology. [Brief explanation of the drawings]

[0047] [Figure 1] Figures 1A-1B show polyacrylamide gel electrophoresis analysis of representative ActRIIB-ECD polypeptide constructs under (Figure 1A) non-reducing and (Figure 1B) reducing conditions. After expression and purification, 1 μg of each protein was loaded onto the gel as indicated: P75: protein 75, P120: protein 120, P121: protein 121, P123: protein 123, P125: protein 125, and P444: protein 444. "NR": non-reducing conditions, "R": reducing conditions. [Figure 2A] Representative results are shown for the benchmark constructs P75 and P444 in a HEK-Blue cell-based assay for inhibition of TGFβ superfamily ligands, as indicated. Figure 2A shows the results for activin A. Error bars indicate the standard error of the mean (SEM). The dashed line represents baseline activity. [Figure 2B]Representative results are shown for the benchmark constructs P75 and P444 in a HEK-Blue cell-based assay for inhibition of TGFβ superfamily ligands, as indicated. Figure 2B shows the results for activin B. Error bars indicate the standard error of the mean (SEM). The dashed line represents baseline activity. [Figure 2C] Representative results are shown for the benchmark constructs P75 and P444 in a HEK-Blue cell-based assay for inhibition of TGFβ superfamily ligands, as indicated. Figure 2C shows the results for GDF-8. Error bars indicate the standard error of the mean (SEM). The dashed line represents baseline activity. [Figure 2D] Representative results are shown for the benchmark constructs P75 and P444 in a HEK-Blue cell-based assay for inhibition of TGFβ superfamily ligands, as indicated. Figure 2D shows the results for GDF-11. Error bars indicate the standard error of the mean (SEM). The dashed line represents baseline activity. [Figure 3A] Representative results are shown for the benchmark constructs P75 and P444 in a HepG2 cell-based assay for inhibition of TGFβ superfamily ligands, as indicated. Figure 3A shows the results for BMP-9. [Figure 3B] Figure 3B shows representative results of the benchmark constructs P75 and P444 in a HepG2 cell-based assay for inhibition of the TGFβ superfamily ligand BMP-10, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 4A] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A against exemplary proteins. Figure 4A shows results for P120 and P121, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 4B]Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A against exemplary proteins. Figure 4B shows the results for P122, P123, and P125, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 4C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A against exemplary proteins. Figure 4C shows results for P126 and P127, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 4D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A against exemplary proteins. Figure 4D shows the results for P622 and P624, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 5A] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B against exemplary proteins. Figure 5A shows results for P120 and P121, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 5B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B against exemplary proteins. Figure 5B shows results for P122, P123, and P125, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 5C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B against exemplary proteins. Figure 5C shows results for P126 and P127, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 5D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B for exemplary proteins. Figure 5D shows the results for P622 and P624, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 6A]Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 6A shows the results for P120 and P121, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 6B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 6B shows the results for P122, P123, and P125, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 6C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 6C shows the results for P126 and P127, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 6D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 6D shows the results for P622 and P624, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 7A] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 7A shows the results for P120 and P121, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 7B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 7B shows the results for P122, P123, and P125, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 7C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 7C shows the results for P126 and P127, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 7D]Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 7D shows the results for P622 and P624, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 8A] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 against exemplary proteins. Figure 8A shows the results for P120 and P121, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 8B] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 against exemplary proteins. Figure 8B shows the results for P122, P123, and P125, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 8C] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 against exemplary proteins. Figure 8C shows the results for P126 and P127, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 8D] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 for exemplary proteins. Figure 8D shows the results for P622 and P624, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 9A] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 9A shows the results for P120 and P121, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 9B] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 9B shows the results for P122, P123, and P125, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 9C]Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 9C shows the results for P126 and P127, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 9D] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 9D shows the results for P622 and P624, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 10A] A comparative chart is shown in which the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) are displayed for exemplary test proteins, as indicated. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a given cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a given cytokine. Figures 10A-10F show exemplary test proteins for which increasing linker length improved potency for all ligands or a subset of ligands. [Figure 10B] 10A-10F show histograms showing the relative % increase or decrease in potency between long and short linkers for each pair of exemplary test proteins. Figures 10A-10F show exemplary test proteins where increasing linker length improved potency for all ligands or a subset of ligands. [Figure 10C] A comparative chart is shown in which the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) are displayed for exemplary test proteins, as indicated. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a given cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a given cytokine. Figures 10A-10F show exemplary test proteins for which increasing linker length improved potency for all ligands or a subset of ligands. [Figure 10D]10A-10F show histograms showing the relative % increase or decrease in potency between long and short linkers for each pair of exemplary test proteins. Figures 10A-10F show exemplary test proteins where increasing linker length improved potency for all ligands or a subset of ligands. [Figure 10E] A comparative chart is shown in which the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) are displayed for exemplary test proteins, as indicated. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a given cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a given cytokine. Figures 10A-10F show exemplary test proteins for which increasing linker length improved potency for all ligands or a subset of ligands. [Figure 10F] 10A-10F show histograms showing the relative % increase or decrease in potency between long and short linkers for each pair of exemplary test proteins. Figures 10A-10F show exemplary test proteins where increasing linker length improved potency for all ligands or a subset of ligands. [Figure 10G] A comparative chart is shown in which the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) are displayed for exemplary test proteins, as indicated. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a given cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a given cytokine. Figures 10G-10J show exemplary test proteins for which increasing linker length decreased potency for a subset of ligands. [Figure 10H] Figures 10G-10J show histograms showing the relative potency increase / decrease (%) between long and short linkers for each pair of exemplary test proteins. Figures 10G-10J show exemplary test proteins for which increasing linker length decreased potency for a subset of ligands. [Figure 10I] A comparative chart is shown in which the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) are displayed for exemplary test proteins, as indicated. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a given cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a given cytokine. Figures 10G-10J show exemplary test proteins for which increasing linker length decreased potency for a subset of ligands. [Figure 10J] Figures 10G-10J show histograms showing the relative potency increase / decrease (%) between long and short linkers for each pair of exemplary test proteins. Figures 10G-10J show exemplary test proteins for which increasing linker length decreased potency for a subset of ligands. [Figure 10K] A comparative chart is shown in which the IC50 values ​​for neutralization of TGFβ superfamily ligands (activin A, activin B, GDF-8, GDF-11, BMP-9, and BMP-10) are displayed for exemplary test proteins, as indicated. Points in the center of the chart indicate low neutralization potency (high IC50 values) for a given cytokine, while points on the edges of the chart indicate high neutralization potency (low IC50 values) for a given cytokine. Figures 10K-10L show exemplary test proteins in which increasing linker length did not affect potency for any of the ligands tested. [Figure 10L] Figures 10K-10L show histograms showing the relative % increase / decrease in potency between long and short linkers for each pair of exemplary test proteins. Figures 10K-10L show exemplary test proteins where increasing linker length did not affect potency for any of the ligands tested. [Figure 10M]As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10N] As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10O]As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10P] As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10Q]As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10R] As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10S]As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10T] As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10U]As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 10V] As shown, for exemplary proteins (Figures 10M-10R) P624, P622, P666, P667, and P1168, (Figures 10S-10U) P1218, P1219, P708, P1220, and P709, and (Figures 10V-10X) P698, P1153, P1154, P701, P1155, and P1156 (Figure 10M, Representative results are shown in HEK-Blue and HepG2 cell-based assays for inhibition of (Figures 10S and 10V) activin A, (Figure 10N) activin B, (Figures 10O, 10T, and 10W) ​​GDF-8, (Figure 10P) GDF-11, (Figures 10Q, 10U, and 10X) BMP-9, and (Figure 10R) BMP-10. Error bars indicate standard error of the mean (SEM). [Figure 11A] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A against exemplary proteins. Figure 11A shows the results for P757, P758, P759, P761, and P762, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 11B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A for exemplary proteins. Figure 11B shows the results for P694, P715, P718, P719, and P720, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 11C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A against exemplary proteins. Figure 11C shows the results for P119, P441, and P124, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 11D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin A for exemplary proteins. Figure 11D shows the results for P687, P1213, P1215, and P1217, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 12A] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B against exemplary proteins. Figure 12A shows the results for P757, P758, P759, P761, and P762, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 12B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B for exemplary proteins. Figure 12B shows the results for P694, P715, P718, P719, and P720, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 12C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B against exemplary proteins. Figure 12C shows the results for P119, P441, and P124. Error bars indicate the standard error of the mean (SEM). [Figure 12D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of activin B for exemplary proteins. Figure 12D shows the results for P687, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 13A]Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 13A shows the results for P757, P758, P759, P761, and P762, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 13B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 13B shows the results for P694, P715, P718, P719, and P720, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 13C] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 13C shows the results for P119, P441, and P124, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 13D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-8 against exemplary proteins. Figure 13D shows the results for P687, P1213, P1215, and P1217, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 14A] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 14A shows the results for P757, P758, P759, P761, and P762, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 14B] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 14B shows the results for P694, P715, P718, P719, and P720, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 14C]Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 14C shows the results for P119, P441, and P124, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 14D] Representative results are shown in a HEK-Blue cell-based assay for the inhibition of GDF-11 against exemplary proteins. Figure 14D shows the results for P687, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 15A] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 for exemplary proteins. Figure 15A shows the results for P757, P758, P759, P761, and P762, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 15B] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 for exemplary proteins. Figure 15B shows the results for P694, P715, P718, P719, and P720, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 15C] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 for exemplary proteins. Figure 15C shows the results for P119, P441, and P124, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 15D] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-9 for exemplary proteins. Figure 15D shows the results for P687, P1213, P1215, and P1217, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 16A] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 16A shows the results for P757, P758, P759, P761, and P762, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 16B] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 16B shows the results for P694, P715, P718, P719, and P720, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 16C] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 16C shows the results for P119, P441, and P124, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 16D] Representative results are shown in a HepG2 cell-based assay for the inhibition of BMP-10 for exemplary proteins. Figure 16D shows the results for P687, as indicated. Error bars indicate the standard error of the mean (SEM). [Figure 17A] Serum concentrations and exposure of P622 and P75 versus dose level are shown. Figure 17A shows the mean concentrations of drug in mouse serum after a single intraperitoneal injection of P121 or P75 at 10 mg / kg and 50 mg / kg. [Figure 17B] Serum concentrations and exposure of P622 and P75 versus dose level are shown. Figure 17B shows the exposure of P622 or P75 versus dose level at 1 mg / kg, 3 mg / kg, 10 mg / kg, 25 mg / kg, and 50 mg / kg in mouse serum. [Figure 18] Figure 1 shows the weight gain in C57BL / 6 mice (n = 6-7 per group) injected twice weekly for 2 weeks with vehicle, P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg). Error bars indicate the standard error of the mean (SEM). [Figure 19A]Figure 19 shows increases in muscle weight and gene expression changes in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Figure 19A shows the results for the gastrocnemius, Figure 19B shows the results for the quadriceps, Figure 19C shows the results for the pectoralis, and Figure 19D shows the results for the triceps. Data are presented as percent increase compared to vehicle at the end of the study, averaged across left and right muscles for each mouse. Figure 19E shows the results for Mss51 expression levels, and Figure 19F evaluates Igf2 expression levels in the quadriceps. After harvesting, tissues were snap-frozen, and <30 mg of tissue was lysed using sonication (5 cycles of 10 seconds, followed by 5 seconds on ice). RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen #74704) according to the manufacturer's instructions. RNA was reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 19B]Figure 19 shows increases in muscle weight and gene expression changes in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Figure 19A shows the results for the gastrocnemius, Figure 19B shows the results for the quadriceps, Figure 19C shows the results for the pectoralis, and Figure 19D shows the results for the triceps. Data are presented as percent increase compared to vehicle at the end of the study, averaged across left and right muscles for each mouse. Figure 19E shows the results for Mss51 expression levels, and Figure 19F evaluates Igf2 expression levels in the quadriceps. After harvesting, tissues were snap-frozen, and <30 mg of tissue was lysed using sonication (5 cycles of 10 seconds, followed by 5 seconds on ice). RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen #74704) according to the manufacturer's instructions. RNA was reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 19C]Figure 19 shows increases in muscle weight and gene expression changes in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Figure 19A shows the results for the gastrocnemius, Figure 19B shows the results for the quadriceps, Figure 19C shows the results for the pectoralis, and Figure 19D shows the results for the triceps. Data are presented as percent increase compared to vehicle at the end of the study, averaged across left and right muscles for each mouse. Figure 19E shows the results for Mss51 expression levels, and Figure 19F evaluates Igf2 expression levels in the quadriceps. After harvesting, tissues were snap-frozen, and <30 mg of tissue was lysed using sonication (5 cycles of 10 seconds, followed by 5 seconds on ice). RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen #74704) according to the manufacturer's instructions. RNA was reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 19D]Figure 19 shows increases in muscle weight and gene expression changes in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Figure 19A shows the results for the gastrocnemius, Figure 19B shows the results for the quadriceps, Figure 19C shows the results for the pectoralis, and Figure 19D shows the results for the triceps. Data are presented as percent increase compared to vehicle at the end of the study, averaged across left and right muscles for each mouse. Figure 19E shows the results for Mss51 expression levels, and Figure 19F evaluates Igf2 expression levels in the quadriceps. After harvesting, tissues were snap-frozen, and <30 mg of tissue was lysed using sonication (5 cycles of 10 seconds, followed by 5 seconds on ice). RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen #74704) according to the manufacturer's instructions. RNA was reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 19E]Figure 19 shows increases in muscle weight and gene expression changes in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Figure 19A shows the results for the gastrocnemius, Figure 19B shows the results for the quadriceps, Figure 19C shows the results for the pectoralis, and Figure 19D shows the results for the triceps. Data are presented as percent increase compared to vehicle at the end of the study, averaged across left and right muscles for each mouse. Figure 19E shows the results for Mss51 expression levels, and Figure 19F evaluates Igf2 expression levels in the quadriceps. After harvesting, tissues were snap-frozen, and <30 mg of tissue was lysed using sonication (5 cycles of 10 seconds, followed by 5 seconds on ice). RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen #74704) according to the manufacturer's instructions. RNA was reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 19F]Figure 19 shows increases in muscle weight and gene expression changes in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Figure 19A shows the results for the gastrocnemius, Figure 19B shows the results for the quadriceps, Figure 19C shows the results for the pectoralis, and Figure 19D shows the results for the triceps. Data are presented as percent increase compared to vehicle at the end of the study, averaged across left and right muscles for each mouse. Figure 19E shows the results for Mss51 expression levels, and Figure 19F evaluates Igf2 expression levels in the quadriceps. After harvesting, tissues were snap-frozen, and <30 mg of tissue was lysed using sonication (5 cycles of 10 seconds, followed by 5 seconds on ice). RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen #74704) according to the manufacturer's instructions. RNA was reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 20] Heart weight normalized by tibia length is shown in C57BL / 6 female mice (n = 6-7 per group, 8-10 weeks old) injected with P75 (16 mg / kg), P622 (4 and 16 mg / kg), and P624 (4 and 16 mg / kg) twice weekly for 2 weeks. Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post-hoc Bonferroni-corrected multiple comparisons test. [Figure 21A]Figures 21A-21C show hematological parameters of female cynomolgus monkeys injected with P622 and P624 at 3 or 30 mg / kg (n=3 per group). Figures 21A-21C show results from P622 treatment. Parameters evaluated were (Figures 21A and 21D) red blood cell (RBC) count, (Figures 21B and 21E) hemoglobin level, and (Figures 21C and 21F) hematocrit. Error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 21B] Figures 21A-21C show hematological parameters of female cynomolgus monkeys injected with P622 and P624 at 3 or 30 mg / kg (n=3 per group). Figures 21A-21C show results from P622 treatment. Parameters evaluated were (Figures 21A and 21D) red blood cell (RBC) count, (Figures 21B and 21E) hemoglobin level, and (Figures 21C and 21F) hematocrit. Error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 21C] Figures 21A-21C show hematological parameters of female cynomolgus monkeys injected with P622 and P624 at 3 or 30 mg / kg (n=3 per group). Figures 21A-21C show results from P622 treatment. Parameters evaluated were (Figures 21A and 21D) red blood cell (RBC) count, (Figures 21B and 21E) hemoglobin level, and (Figures 21C and 21F) hematocrit. Error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 21D]Figures 21D-21F show hematological parameters of female cynomolgus monkeys injected with P622 and P624 at 3 or 30 mg / kg (n=3 per group). Results from P624 treatment are shown. Parameters evaluated were (Figures 21A and 21D) red blood cell (RBC) count, (Figures 21B and 21E) hemoglobin level, and (Figures 21C and 21F) hematocrit. Error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 21E] Figures 21D-21F show hematological parameters of female cynomolgus monkeys injected with P622 and P624 at 3 or 30 mg / kg (n=3 per group). Results from P624 treatment are shown. Parameters evaluated were (Figures 21A and 21D) red blood cell (RBC) count, (Figures 21B and 21E) hemoglobin level, and (Figures 21C and 21F) hematocrit. Error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 21F] Figures 21D-21F show hematological parameters of female cynomolgus monkeys injected with P622 and P624 at 3 or 30 mg / kg (n=3 per group). Results from P624 treatment are shown. Parameters evaluated were (Figures 21A and 21D) red blood cell (RBC) count, (Figures 21B and 21E) hemoglobin level, and (Figures 21C and 21F) hematocrit. Error bars indicate the standard error of the mean (SEM). Results were analyzed by two-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 22A] Representative results are shown for the HEK-Blue cell-based assay for the inhibition of activin A (FIG. 22A), activin B (FIG. 22B), GDF-8 (FIG. 22C), and GDF-11 (FIG. 22D) and for the inhibition of BMP-9 (FIG. 22E) and BMP-10 (FIG. 22F) against exemplary proteins P670, P671, and P674. Error bars indicate the standard error of the mean (SEM). [Figure 22B] Representative results are shown for the HEK-Blue cell-based assay for the inhibition of activin A (FIG. 22A), activin B (FIG. 22B), GDF-8 (FIG. 22C), and GDF-11 (FIG. 22D) and for the inhibition of BMP-9 (FIG. 22E) and BMP-10 (FIG. 22F) against exemplary proteins P670, P671, and P674. Error bars indicate the standard error of the mean (SEM). [Figure 22C] Representative results are shown for the HEK-Blue cell-based assay for the inhibition of activin A (FIG. 22A), activin B (FIG. 22B), GDF-8 (FIG. 22C), and GDF-11 (FIG. 22D) and for the inhibition of BMP-9 (FIG. 22E) and BMP-10 (FIG. 22F) against exemplary proteins P670, P671, and P674. Error bars indicate the standard error of the mean (SEM). [Figure 22D] Representative results are shown for the HEK-Blue cell-based assay for the inhibition of activin A (FIG. 22A), activin B (FIG. 22B), GDF-8 (FIG. 22C), and GDF-11 (FIG. 22D) and for the inhibition of BMP-9 (FIG. 22E) and BMP-10 (FIG. 22F) against exemplary proteins P670, P671, and P674. Error bars indicate the standard error of the mean (SEM). [Figure 22E] Representative results are shown for the HEK-Blue cell-based assay for the inhibition of activin A (FIG. 22A), activin B (FIG. 22B), GDF-8 (FIG. 22C), and GDF-11 (FIG. 22D) and for the inhibition of BMP-9 (FIG. 22E) and BMP-10 (FIG. 22F) against exemplary proteins P670, P671, and P674. Error bars indicate the standard error of the mean (SEM). [Figure 22F]Representative results are shown for the HEK-Blue cell-based assay for the inhibition of activin A (FIG. 22A), activin B (FIG. 22B), GDF-8 (FIG. 22C), and GDF-11 (FIG. 22D) and for the inhibition of BMP-9 (FIG. 22E) and BMP-10 (FIG. 22F) against exemplary proteins P670, P671, and P674. Error bars indicate the standard error of the mean (SEM). [Figure 23A] The efficacy of P670, P671, and P674 in a rat PAH model is shown. Rats were injected with monocrotaline (MCT; 60 mg / kg) on ​​day 0, followed by biweekly injections of P670, P671, and P674 (1, 4, or 16 mg / kg) starting on day 1. On day 29, animals were sacrificed, and the following parameters were assessed: (Figure 23A) Fulton index (ratio of right ventricular mass to left ventricular and septal mass [RV / LV+S]), (Figure 23B) mean pulmonary artery pressure (mPAP), (Figure 23C) right ventricular systolic pressure (RVSP), (Figure 23D) right ventricular free wall thickness (RVFWT), (Figure 23E) velocity time integral (VTI), and (Figure 23F) pulmonary artery acceleration time (PAAT). Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 23B] The efficacy of P670, P671, and P674 in a rat PAH model is shown. Rats were injected with monocrotaline (MCT; 60 mg / kg) on ​​day 0, followed by biweekly injections of P670, P671, and P674 (1, 4, or 16 mg / kg) starting on day 1. On day 29, animals were sacrificed, and the following parameters were assessed: (Figure 23A) Fulton index (ratio of right ventricular mass to left ventricular and septal mass [RV / LV+S]), (Figure 23B) mean pulmonary artery pressure (mPAP), (Figure 23C) right ventricular systolic pressure (RVSP), (Figure 23D) right ventricular free wall thickness (RVFWT), (Figure 23E) velocity time integral (VTI), and (Figure 23F) pulmonary artery acceleration time (PAAT). Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 23C]The efficacy of P670, P671, and P674 in a rat PAH model is shown. Rats were injected with monocrotaline (MCT; 60 mg / kg) on ​​day 0, followed by biweekly injections of P670, P671, and P674 (1, 4, or 16 mg / kg) starting on day 1. On day 29, animals were sacrificed, and the following parameters were assessed: (Figure 23A) Fulton index (ratio of right ventricular mass to left ventricular and septal mass [RV / LV+S]), (Figure 23B) mean pulmonary artery pressure (mPAP), (Figure 23C) right ventricular systolic pressure (RVSP), (Figure 23D) right ventricular free wall thickness (RVFWT), (Figure 23E) velocity time integral (VTI), and (Figure 23F) pulmonary artery acceleration time (PAAT). Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 23D] The efficacy of P670, P671, and P674 in a rat PAH model is shown. Rats were injected with monocrotaline (MCT; 60 mg / kg) on ​​day 0, followed by biweekly injections of P670, P671, and P674 (1, 4, or 16 mg / kg) starting on day 1. On day 29, animals were sacrificed, and the following parameters were assessed: (Figure 23A) Fulton index (ratio of right ventricular mass to left ventricular and septal mass [RV / LV+S]), (Figure 23B) mean pulmonary artery pressure (mPAP), (Figure 23C) right ventricular systolic pressure (RVSP), (Figure 23D) right ventricular free wall thickness (RVFWT), (Figure 23E) velocity time integral (VTI), and (Figure 23F) pulmonary artery acceleration time (PAAT). Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 23E]The efficacy of P670, P671, and P674 in a rat PAH model is shown. Rats were injected with monocrotaline (MCT; 60 mg / kg) on ​​day 0, followed by biweekly injections of P670, P671, and P674 (1, 4, or 16 mg / kg) starting on day 1. On day 29, animals were sacrificed, and the following parameters were assessed: (Figure 23A) Fulton index (ratio of right ventricular mass to left ventricular and septal mass [RV / LV+S]), (Figure 23B) mean pulmonary artery pressure (mPAP), (Figure 23C) right ventricular systolic pressure (RVSP), (Figure 23D) right ventricular free wall thickness (RVFWT), (Figure 23E) velocity time integral (VTI), and (Figure 23F) pulmonary artery acceleration time (PAAT). Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 23F] The efficacy of P670, P671, and P674 in a rat PAH model is shown. Rats were injected with monocrotaline (MCT; 60 mg / kg) on ​​day 0, followed by biweekly injections of P670, P671, and P674 (1, 4, or 16 mg / kg) starting on day 1. On day 29, animals were sacrificed, and the following parameters were assessed: (Figure 23A) Fulton index (ratio of right ventricular mass to left ventricular and septal mass [RV / LV+S]), (Figure 23B) mean pulmonary artery pressure (mPAP), (Figure 23C) right ventricular systolic pressure (RVSP), (Figure 23D) right ventricular free wall thickness (RVFWT), (Figure 23E) velocity time integral (VTI), and (Figure 23F) pulmonary artery acceleration time (PAAT). Error bars indicate standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 24A]Figure 24 shows the efficacy of P670, P671, and P674 in a rat PAH model. Using the 16 mg / kg data from Figure 23 for each drug, rats were divided into subgroups based on their exposure to drug between days 7 and 29. T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. The parameters evaluated were (Figure 24A) Fulton's index, (Figure 24B) mPAP, (Figure 24C) RVSP, (Figure 24D) RVFWT, (Figure 24E) VTI, and (Figure 24F) PAAT. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 24B] Figure 24 shows the efficacy of P670, P671, and P674 in a rat PAH model. Using the 16 mg / kg data from Figure 23 for each drug, rats were divided into subgroups based on their exposure to drug between days 7 and 29. T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. The parameters evaluated were (Figure 24A) Fulton's index, (Figure 24B) mPAP, (Figure 24C) RVSP, (Figure 24D) RVFWT, (Figure 24E) VTI, and (Figure 24F) PAAT. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 24C] Figure 24 shows the efficacy of P670, P671, and P674 in a rat PAH model. Using the 16 mg / kg data from Figure 23 for each drug, rats were divided into subgroups based on their exposure to drug between days 7 and 29. T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. The parameters evaluated were (Figure 24A) Fulton's index, (Figure 24B) mPAP, (Figure 24C) RVSP, (Figure 24D) RVFWT, (Figure 24E) VTI, and (Figure 24F) PAAT. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 24D]Figure 24 shows the efficacy of P670, P671, and P674 in a rat PAH model. Using the 16 mg / kg data from Figure 23 for each drug, rats were divided into subgroups based on their exposure to drug between days 7 and 29. T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. The parameters evaluated were (Figure 24A) Fulton's index, (Figure 24B) mPAP, (Figure 24C) RVSP, (Figure 24D) RVFWT, (Figure 24E) VTI, and (Figure 24F) PAAT. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 24E] Figure 24 shows the efficacy of P670, P671, and P674 in a rat PAH model. Using the 16 mg / kg data from Figure 23 for each drug, rats were divided into subgroups based on their exposure to drug between days 7 and 29. T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. The parameters evaluated were (Figure 24A) Fulton's index, (Figure 24B) mPAP, (Figure 24C) RVSP, (Figure 24D) RVFWT, (Figure 24E) VTI, and (Figure 24F) PAAT. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 24F] Figure 24 shows the efficacy of P670, P671, and P674 in a rat PAH model. Using the 16 mg / kg data from Figure 23 for each drug, rats were divided into subgroups based on their exposure to drug between days 7 and 29. T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. The parameters evaluated were (Figure 24A) Fulton's index, (Figure 24B) mPAP, (Figure 24C) RVSP, (Figure 24D) RVFWT, (Figure 24E) VTI, and (Figure 24F) PAAT. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 25A]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25A-B). Pathological evaluation of stained sections was performed for each animal by characterizing 30 vessels with an external diameter of 10–50 μm within the peripheral region as non-muscularized, partially muscularized, or fully muscularized. [Figure 25B] Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25A-B). Pathological evaluation of stained sections was performed for each animal by characterizing 30 vessels with an external diameter of 10–50 μm within the peripheral region as non-muscularized, partially muscularized, or fully muscularized. [Figure 25C] Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25C-25D) Medial and outer wall diameters were measured from muscularized vessels, and the mean percent luminal obstruction and wall thickness were calculated for each animal (medial wall thickness index, MTI). [Figure 25D]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25C-25D) Medial and outer wall diameters were measured from muscularized vessels, and the mean percent luminal obstruction and wall thickness were calculated for each animal (medial wall thickness index, MTI). [Figure 25E] Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25E-F) Each animal was also assigned a score based on fibrin levels in the interstitium, alveolar hemorrhage, and cellular infiltration, referred to herein as the total histopathology score. [Figure 25F] Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25E-F) Each animal was also assigned a score based on fibrin levels in the interstitium, alveolar hemorrhage, and cellular infiltration, referred to herein as the total histopathology score. [Figure 25G]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25G-K). Expression levels of Inhba and Ctgf in the lung (Figures 25G-H) and Nppb, Ctgf, and Nppa in the right ventricle (Figures 25I-K) were assessed. After collection, tissues were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Rpl13a, and B2m were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 25H]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25G-K). Expression levels of Inhba and Ctgf in the lung (Figures 25G-H) and Nppb, Ctgf, and Nppa in the right ventricle (Figures 25I-K) were assessed. After collection, tissues were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Rpl13a, and B2m were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 25I]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25G-K). Expression levels of Inhba and Ctgf in the lung (Figures 25G-H) and Nppb, Ctgf, and Nppa in the right ventricle (Figures 25I-K) were assessed. After collection, tissues were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Rpl13a, and B2m were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 25J]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25G-K). Expression levels of Inhba and Ctgf in the lung (Figures 25G-H) and Nppb, Ctgf, and Nppa in the right ventricle (Figures 25I-K) were assessed. After collection, tissues were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Rpl13a, and B2m were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 25K]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E (Figures 25G-K). Expression levels of Inhba and Ctgf in the lung (Figures 25G-H) and Nppb, Ctgf, and Nppa in the right ventricle (Figures 25I-K) were assessed. After collection, tissues were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Rpl13a, and B2m were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 25L] Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25L-O) RV samples from three animals per group (naive, MCT + vehicle, MCT + P670 16 mg / kg, and MCT + P671 16 mg / kg) were analyzed by RNA sequencing. (Figure 25L-M) Expression level heatmap for all differentially expressed genes (DEGs) between naive and MCT samples (Figure 25L) and for DEGs belonging to KEGG pathways of interest in this disease model (Figure 25M). Values ​​are centered around the mean expression level in naive animals. [Figure 25M]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25L-O) RV samples from three animals per group (naive, MCT + vehicle, MCT + P670 16 mg / kg, and MCT + P671 16 mg / kg) were analyzed by RNA sequencing. (Figure 25L-M) Expression level heatmap for all differentially expressed genes (DEGs) between naive and MCT samples (Figure 25L) and for DEGs belonging to KEGG pathways of interest in this disease model (Figure 25M). Values ​​are centered around the mean expression level in naive animals. [Figure 25N] Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25L-O) RV samples from three animals per group (naive, MCT + vehicle, MCT + P670 16 mg / kg, and MCT + P671 16 mg / kg) were analyzed by RNA sequencing. (Figures 25N-O) The number of DEGs was determined using the naive group (Figure 25N) or the MCT group (Figure 25O) as a reference. [Figure 25O]Figure 25 shows the efficacy of 16 mg / kg P670, P671, and P674 in a rat PAH model. Rats were divided into subgroups based on drug exposure between days 7 and 29: T1: lowest exposure level, T2: intermediate exposure level, and T3: highest exposure level. For further histological analysis, lungs were harvested on day 29, inflated, and perfused with 10% neutral buffered formalin. After paraffin embedding, sections were cut and stained with H&E. (Figures 25L-O) RV samples from three animals per group (naive, MCT + vehicle, MCT + P670 16 mg / kg, and MCT + P671 16 mg / kg) were analyzed by RNA sequencing. (Figures 25L-25M) Expression level heatmaps for all differentially expressed genes (DEGs) between naive and MCT samples (Figure 25L) and for DEGs belonging to KEGG pathways of interest in this disease model (Figure 25M). Values ​​are centered around the mean expression level of naive animals. (Figures 25N-25O) Number of DEGs using the naive group (Figure 25N) or the MCT group (Figure 25O) as reference. [Figure 26A] Figure 26 shows the efficacy of P444, P622, and P624 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokines (50 ng / mL, except for 25 ng / mL activin AB). (Figure 26A) activin A (n=5), (Figure 26B) GDF-8 (n=5), (Figure 26C) GDF-11 (n=6), and (Figure 26D) activin AB (n=1). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 and CTGF mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 26B]Figure 26 shows the efficacy of P444, P622, and P624 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokines (50 ng / mL, except for 25 ng / mL activin AB). (Figure 26A) activin A (n=5), (Figure 26B) GDF-8 (n=5), (Figure 26C) GDF-11 (n=6), and (Figure 26D) activin AB (n=1). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 and CTGF mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 26C] Figure 26 shows the efficacy of P444, P622, and P624 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokines (50 ng / mL, except for 25 ng / mL activin AB). (Figure 26A) activin A (n=5), (Figure 26B) GDF-8 (n=5), (Figure 26C) GDF-11 (n=6), and (Figure 26D) activin AB (n=1). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 and CTGF mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 26D]Figure 26 shows the efficacy of P444, P622, and P624 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokines (50 ng / mL, except for 25 ng / mL activin AB). (Figure 26A) activin A (n=5), (Figure 26B) GDF-8 (n=5), (Figure 26C) GDF-11 (n=6), and (Figure 26D) activin AB (n=1). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 and CTGF mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by post hoc Bonferroni-corrected multiple comparison tests. [Figure 27A] Figure 27 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 27A) Activin A + GDF-8 (n = 3), and (Figure 27B) Activin A + GDF-11 (n = 3). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 27A and 27D), CTGF (Figures 27B and 27E), and INHBA (Figures 27C and 27F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 27B]Figure 27 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 27A) Activin A + GDF-8 (n = 3), and (Figure 27B) Activin A + GDF-11 (n = 3). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 27A and 27D), CTGF (Figures 27B and 27E), and INHBA (Figures 27C and 27F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 27C] Figure 27 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 27A) Activin A + GDF-8 (n = 3), and (Figure 27B) Activin A + GDF-11 (n = 3). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 27A and 27D), CTGF (Figures 27B and 27E), and INHBA (Figures 27C and 27F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 27D]Figure 27 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 27A) Activin A + GDF-8 (n = 3), and (Figure 27B) Activin A + GDF-11 (n = 3). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 27A and 27D), CTGF (Figures 27B and 27E), and INHBA (Figures 27C and 27F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 27E] Figure 27 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 27A) Activin A + GDF-8 (n = 3), and (Figure 27B) Activin A + GDF-11 (n = 3). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 27A and 27D), CTGF (Figures 27B and 27E), and INHBA (Figures 27C and 27F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 27F]Figure 27 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 27A) Activin A + GDF-8 (n = 3), and (Figure 27B) Activin A + GDF-11 (n = 3). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 27A and 27D), CTGF (Figures 27B and 27E), and INHBA (Figures 27C and 27F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 28A] Figure 28 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 28A) GDF-8 + GDF-11 (n = 3), and (Figure 28B) activin B + GDF-8 (n = 2). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 28A and 28D), CTGF (Figures 28B and 28E), and INHBA (Figures 28C and 28F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 28B]Figure 28 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 28A) GDF-8 + GDF-11 (n = 3), and (Figure 28B) activin B + GDF-8 (n = 2). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 28A and 28D), CTGF (Figures 28B and 28E), and INHBA (Figures 28C and 28F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 28C] Figure 28 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 28A) GDF-8 + GDF-11 (n = 3), and (Figure 28B) activin B + GDF-8 (n = 2). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 28A and 28D), CTGF (Figures 28B and 28E), and INHBA (Figures 28C and 28F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 28D]Figure 28 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 28A) GDF-8 + GDF-11 (n = 3), and (Figure 28B) activin B + GDF-8 (n = 2). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 28A and 28D), CTGF (Figures 28B and 28E), and INHBA (Figures 28C and 28F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 28E] Figure 28 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 28A) GDF-8 + GDF-11 (n = 3), and (Figure 28B) activin B + GDF-8 (n = 2). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 28A and 28D), CTGF (Figures 28B and 28E), and INHBA (Figures 28C and 28F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 28F]Figure 28 shows the efficacy of P444 and P622 in primary human pulmonary artery smooth muscle cells (PASMC). On day 1, 50,000 cells were seeded in growth medium in a 96-well plate. On day 2, exemplary agents (3.7 or 33.3 nM) were incubated with the cells in serum-free medium for 30 minutes, followed by the addition of the relevant cytokine combination (50 ng / mL per cytokine). (Figure 28A) GDF-8 + GDF-11 (n = 3), and (Figure 28B) activin B + GDF-8 (n = 2). On day 3 (approximately 24 hours later), cells were harvested and RNA was collected for RT-qPCR analysis. ACTA2 (Figures 28A and 28D), CTGF (Figures 28B and 28E), and INHBA (Figures 28C and 28F) mRNA levels were assessed relative to the housekeeping gene GAPDH. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 29A] Figure 29 shows the in vivo efficacy of exemplary agents P75, P121, P444, P622, and P624 (30 and / or 50 mg / kg). Male mice aged 6 to 8 weeks were injected with exemplary agents, and lungs were harvested 4 days later and fixed in neutral buffered formalin. (Figure 29A) Phosphorylated SMAD2 (pSMAD2) and (Figure 29B) pSMAD3 were assessed by immunohistology. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. (Figure 29C) Representative pSMAD3 images from certain exemplary agents are displayed. [Figure 29B]Figure 29 shows the in vivo efficacy of exemplary agents P75, P121, P444, P622, and P624 (30 and / or 50 mg / kg). Male mice aged 6 to 8 weeks were injected with exemplary agents, and lungs were harvested 4 days later and fixed in neutral buffered formalin. (Figure 29A) Phosphorylated SMAD2 (pSMAD2) and (Figure 29B) pSMAD3 were assessed by immunohistology. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. (Figure 29C) Representative pSMAD3 images from certain exemplary agents are displayed. [Figure 29C] Figure 29 shows the in vivo efficacy of exemplary agents P75, P121, P444, P622, and P624 (30 and / or 50 mg / kg). Male mice aged 6 to 8 weeks were injected with exemplary agents, and lungs were harvested 4 days later and fixed in neutral buffered formalin. (Figure 29A) Phosphorylated SMAD2 (pSMAD2) and (Figure 29B) pSMAD3 were assessed by immunohistology. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. (Figure 29C) Representative pSMAD3 images from certain exemplary agents are displayed. [Figure 30A] Figure 30 shows the efficacy of exemplary agents P75, P444, P622, and P624 (30 mg / kg) in vivo. Male mice aged 6 to 8 weeks were injected with exemplary agents, and lungs were collected 4 days later, and RNA was extracted for RT-qPCR analysis. (Figure 30A) Inhba, (Figure 30B) Gdf11, and (Figure 30C) Serpine1 mRNA levels were assessed relative to the housekeeping genes Rpl13a, Rpl19, Gusb, and Gapdh. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 30B]Figure 30 shows the efficacy of exemplary agents P75, P444, P622, and P624 (30 mg / kg) in vivo. Male mice aged 6 to 8 weeks were injected with exemplary agents, and lungs were collected 4 days later, and RNA was extracted for RT-qPCR analysis. (Figure 30A) Inhba, (Figure 30B) Gdf11, and (Figure 30C) Serpine1 mRNA levels were assessed relative to the housekeeping genes Rpl13a, Rpl19, Gusb, and Gapdh. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 30C] Figure 30 shows the efficacy of exemplary agents P75, P444, P622, and P624 (30 mg / kg) in vivo. Male mice aged 6 to 8 weeks were injected with exemplary agents, and lungs were collected 4 days later, and RNA was extracted for RT-qPCR analysis. (Figure 30A) Inhba, (Figure 30B) Gdf11, and (Figure 30C) Serpine1 mRNA levels were assessed relative to the housekeeping genes Rpl13a, Rpl19, Gusb, and Gapdh. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. [Figure 31A]Figure 31 shows the in vivo efficacy of exemplary agents P75, P444, P622, and P624 (1, 3, 10, 25, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and body weights, organs, and plasma were collected 21 days later. (Figure 31A) Body weights on day 21 normalized to the vehicle group. (Figures 31B-C) Gastrocnemius and tibialis anterior weights were normalized to the vehicle group and plotted as a function of dose. (Figure 31D) FSH levels were assessed in plasma on day 21 (MPTMAG-49K, Millipore Sigma). (Figure 31E) FSHβ (Fshb) gene expression levels in the pituitary gland were assessed. Pituitaries were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 31B]Figure 31 shows the in vivo efficacy of exemplary agents P75, P444, P622, and P624 (1, 3, 10, 25, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and body weights, organs, and plasma were collected 21 days later. (Figure 31A) Body weights on day 21 normalized to the vehicle group. (Figures 31B-C) Gastrocnemius and tibialis anterior weights were normalized to the vehicle group and plotted as a function of dose. (Figure 31D) FSH levels were assessed in plasma on day 21 (MPTMAG-49K, Millipore Sigma). (Figure 31E) FSHβ (Fshb) gene expression levels in the pituitary gland were assessed. Pituitaries were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 31C]Figure 31 shows the in vivo efficacy of exemplary agents P75, P444, P622, and P624 (1, 3, 10, 25, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and body weights, organs, and plasma were collected 21 days later. (Figure 31A) Body weights on day 21 normalized to the vehicle group. (Figures 31B-C) Gastrocnemius and tibialis anterior weights were normalized to the vehicle group and plotted as a function of dose. (Figure 31D) FSH levels were assessed in plasma on day 21 (MPTMAG-49K, Millipore Sigma). (Figure 31E) FSHβ (Fshb) gene expression levels in the pituitary gland were assessed. Pituitaries were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 31D]Figure 31 shows the in vivo efficacy of exemplary agents P75, P444, P622, and P624 (1, 3, 10, 25, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and body weights, organs, and plasma were collected 21 days later. (Figure 31A) Body weights on day 21 normalized to the vehicle group. (Figures 31B-C) Gastrocnemius and tibialis anterior weights were normalized to the vehicle group and plotted as a function of dose. (Figure 31D) FSH levels were assessed in plasma on day 21 (MPTMAG-49K, Millipore Sigma). (Figure 31E) FSHβ (Fshb) gene expression levels in the pituitary gland were assessed. Pituitaries were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 31E]Figure 31 shows the in vivo efficacy of exemplary agents P75, P444, P622, and P624 (1, 3, 10, 25, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and body weights, organs, and plasma were collected 21 days later. (Figure 31A) Body weights on day 21 normalized to the vehicle group. (Figures 31B-C) Gastrocnemius and tibialis anterior weights were normalized to the vehicle group and plotted as a function of dose. (Figure 31D) FSH levels were assessed in plasma on day 21 (MPTMAG-49K, Millipore Sigma). (Figure 31E) FSHβ (Fshb) gene expression levels in the pituitary gland were assessed. Pituitaries were snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted and reverse transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb and Gapdh were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 32A] The efficacy of exemplary drugs P75, P444, and P622 in vivo in relation to each animal's exposure is shown. (A-B) The data in Figures 31D-E were plotted against each animal's exposure (area under the curve [AUC]), and linear or nonlinear fits were plotted using GraphPad Prism 9.0. [Figure 32B] The efficacy of exemplary drugs P75, P444, and P622 in vivo in relation to each animal's exposure is shown. (A-B) The data in Figures 31D-E were plotted against each animal's exposure (area under the curve [AUC]), and linear or nonlinear fits were plotted using GraphPad Prism 9.0. [Figure 33A]Figure 1 shows the in vivo efficacy of the exemplary agents P444 and P622 (1, 10, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and muscle tissue was collected 21 days later. (A-B) Mss51 and Igf2 expression levels in the right gastrocnemius muscle. After collection, tissue was snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted, reverse-transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Gapdh, and Rpl13 were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Figure 33B] Figure 1 shows the in vivo efficacy of the exemplary agents P444 and P622 (1, 10, and 50 mg / kg). Wild-type C57BL / 6 male mice were injected (subcutaneously) twice weekly with vehicle or exemplary agents, and muscle tissue was collected 21 days later. (A-B) Mss51 and Igf2 expression levels in the right gastrocnemius muscle. After collection, tissue was snap-frozen and lysed using a gentleMACS™ Octo Dissociator (Miltenyi Biotech). RNA was extracted, reverse-transcribed, and gene expression was assessed by qPCR according to the manufacturer's instructions (Qiagen). Actb, Gapdh, and Rpl13 were used as housekeeping genes. Error bars indicate the standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by a post-hoc Bonferroni-corrected multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Figure 34] The efficacy of exemplary drugs P444 and P622 in vivo in relation to exposure for each animal is shown. The data in Figures 33A-33B were plotted against exposure for each animal (area under the curve [AUC]), and linear or nonlinear fits were plotted using GraphPad Prism 9.0. [Figure 35A]Volcano plots of genes associated with eight KEGG pathways selected for their relevance to PH pathophysiology and their expression levels in the RV compared to the MCT of naive animals are shown (see Figure 35A). Results of gene expression changes after treatment with exemplary drugs P671, P674, and P670 are shown in Figures 35B, 35C, and 35D, respectively. All genes are plotted as a function of their fold-change expression (down- or up-regulated as log2 fold change) and statistical significance (as -log10 of adjusted p-value). Exemplary genes of interest related to the TGFβ signaling pathway and associated with heart failure are highlighted for each graph. The horizontal dashed line indicates a significance level of p=0.05. [Figure 35B] Volcano plots of genes associated with eight KEGG pathways selected for their relevance to PH pathophysiology and their expression levels in the RV compared to the MCT of naive animals are shown (see Figure 35A). Results of gene expression changes after treatment with exemplary drugs P671, P674, and P670 are shown in Figures 35B, 35C, and 35D, respectively. All genes are plotted as a function of their fold-change expression (down- or up-regulated as log2 fold change) and statistical significance (as -log10 of adjusted p-value). Exemplary genes of interest related to the TGFβ signaling pathway and associated with heart failure are highlighted for each graph. The horizontal dashed line indicates a significance level of p=0.05. [Figure 35C]Volcano plots of genes associated with eight KEGG pathways selected for their relevance to PH pathophysiology and their expression levels in the RV compared to the MCT of naive animals are shown (see Figure 35A). Results of gene expression changes after treatment with exemplary drugs P671, P674, and P670 are shown in Figures 35B, 35C, and 35D, respectively. All genes are plotted as a function of their fold-change expression (down- or up-regulated as log2 fold change) and statistical significance (as -log10 of adjusted p-value). Exemplary genes of interest related to the TGFβ signaling pathway and associated with heart failure are highlighted for each graph. The horizontal dashed line indicates a significance level of p=0.05. [Figure 35D] Volcano plots of genes associated with eight KEGG pathways selected for their relevance to PH pathophysiology and their expression levels in the RV compared to the MCT of naive animals are shown (see Figure 35A). Results of gene expression changes after treatment with exemplary drugs P671, P674, and P670 are shown in Figures 35B, 35C, and 35D, respectively. All genes are plotted as a function of their fold-change expression (down- or up-regulated as log2 fold change) and statistical significance (as -log10 of adjusted p-value). Exemplary genes of interest related to the TGFβ signaling pathway and associated with heart failure are highlighted for each graph. The horizontal dashed line indicates a significance level of p=0.05. [Figure 36] 1 provides an exemplary schematic of a binder described herein. DETAILED DESCRIPTION OF THE INVENTION

[0048] overview Activin type II receptor is a single transmembrane domain receptor that regulates the signal of ligands in the TGFβ superfamily. There are two types of activin type II receptors: ActRIIA and ActRIIB. Examples of TGFβ superfamily ligands include activins (e.g., activin A and activin B), inhibins, growth differentiation factors (GDFs) (e.g., GDF-8, also known as myostatin and GDF-11), and bone morphogenetic proteins (BMPs) (BMP-9, BMP-10). The activity of TGFβ superfamily ligands is involved in various diseases and disorders, including pulmonary hypertension (PH), fibrosis, muscle diseases (including muscular dystrophy), metabolic disorders (including type II diabetes), bone diseases, and anemia.

[0049] One approach to developing therapeutics that inhibit TGFβ superfamily ligand function has been to use soluble decoy receptors (also called receptor ectodomain (ECD)-based ligand traps) to bind and sequester the ligand, thereby blocking 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 approaches. For example, polypeptide fusions based on the TGFβ receptor ectodomain that bind to or "trap" TGFβ1 and / or TGFβ2 and / or TGFβ3 ligand isoforms have been used to inhibit TGFβ signaling (e.g., WO01 / 83525, WO2005 / 028517, WO2008 / 113185, WO2008 / 157367, WO2010 / 0031168, WO2010 / 0031168). (See US2005 / 0203022, US2007 / 0244042, US8318135, US8658135, US8815247, US2015 / 0225483, US2015 / 0056199, and WO2017 / 037634).

[0050] In the pulmonary endothelium and vasculature, bone morphogenetic proteins (BMPs) can induce antiproliferative effects on smooth muscle cell (SMC) and endothelial cell (EC) survival, whereas activins and growth differentiation factors (GDFs) can induce opposing effects, namely, pro-proliferative effects on SMCs and apoptosis of ECs (Yung, LM et al., 2020; Ryanto, GR et al., 2021). Under physiological conditions, these ligands act in concert to maintain homeostasis. However, in certain disease states, such as PAH, these pathways become imbalanced. For example, approximately 80% of familial cases and approximately 20% of idiopathic cases of PAH are caused by mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) (Austin, ED and Loyd, JE, 2007; Quarck, R. and Perros, F., 2017). This leads to an imbalance between the activin / GDF and BMP signaling pathways (Ryanto, GRT et al., 2021). Therefore, it is desirable to provide a receptor ectodomain-based trap that can neutralize certain ligands but not others to rebalance the pathways and reestablish vascular homeostasis.

[0051] Some ECD-based traps contain amino acid mutations in the ectodomain portion of the compound to alter binding to one or more TGFβ superfamily ligands. See, for example, WO2021 / 158675, WO2022 / 150590, WO2021 / 158675, WO2022 / 072882, WO2021 / 189019, and WO2021 / 189010. While point mutations in the ActRIIB ECD have been described in the context of other trap-based drugs, this disclosure highlights the effects of these mutations when combined with long peptide linkers. Specifically, the data in this application demonstrate that certain mutations in the ActRIIB ECD have unpredictable effects on TGFβ superfamily ligand binding when combined with long peptide linkers (i.e., 10 or more amino acids).

[0052] Thus, the present application provides TGFβ superfamily ligand binders that exhibit improved ligand-binding profiles and therapeutic efficacy. The combination of point mutations in the ActRIIB ECD with long linkers described herein provides a platform where the beneficial ligand-binding profiles of these point mutations can be further enhanced by combining them with linkers of specific lengths. These platform compounds are useful for the treatment of a variety of diseases and disorders driven by TGFβ superfamily ligands, including pulmonary hypertension, muscle diseases, metabolic disorders, bone diseases, anemia, and fibrosis.

[0053] The present technology is described in more detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology can be implemented or all the features that can be added to the technology. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be omitted from that embodiment. Additionally, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, and such modifications and additions do not depart from the present technology. Thus, the following description is intended to illustrate some specific embodiments of the present technology, but is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0054] definition In order to provide a clear and consistent understanding of the terms used herein, certain definitions are provided below. Furthermore, 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.

[0055] The use of the terms "a," "an," and "the," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Similarly, the term "another" can mean at least a second or more. These terms should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0056] As used herein, the terms "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "include" and "includes"), or "containing" (and any form of "containing", such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. The term "consisting of" is to be construed as close-ended.

[0057] The term "about" is used to indicate that a value or amount refers to the actual given value and approximations of such a given value that would be reasonably inferred based on common skill in the art, including equivalents and approximations based on experimental and / or measurement conditions for such a given value. For example, the term "about" in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0058] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of the specified features or components, regardless of the presence or absence of the others. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. The term "or," as used herein, is understood to be inclusive and encompasses both "or" and "and," unless specifically stated otherwise or clear from the context. For example, an embodiment of "a composition comprising A or B" would typically present an aspect having a composition containing both A and B. However, "or" should be interpreted to exclude those aspects that cannot be combined without contradiction (e.g., a composition pH of 9-10 or 7-8).

[0059] As used herein, terms such as "1 to 20" should be understood to include any individual value subsumed therein, inclusive of 1 and 20. Thus, the term "1 to 20" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20. Terms such as "1 to 20" also include any individual subranges subsumed therein, inclusive of 1 and 20. Thus, the term "1 to 20" also includes subranges such as "1 to 9," "2 to 9," "3 to 5," "5 to 9," "5 to 20," "8 to 20," etc. The same applies to similar expressions such as, but not limited to, "1 to 19," "1 to 18," "1 to 10," "1 to 9," "5 to 15," etc.

[0060] As used herein, terms such as "about 15 to about 35" should be understood to include any individual value between 15 and 35, inclusive. Thus, terms such as "about 15 to about 35" include any number between 15 and 35, inclusive, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and / or 35. Terms such as "about 15 to about 35" also include any individual subranges between 15 and 35, inclusive, such as "about 16 to about 34," "about 16 to about 24," "about 24 to about 34," etc. The term "about" in the context of a number of amino acids means that the particular number of amino acids is specifically included, allowing for a + / - 2 variation in the number of amino acid residues. Thus, terms such as "about 15 to about 35" also include "13 to 37," "13 to 35," "17 to 37," "17 to 35," etc. The same applies to similar expressions such as, but not limited to, "about 16 to about 34," "about 16 to about 24," and "about 24 to about 34."

[0061] As used herein, terms such as "at least 80% identical" are understood to include 80% and 100%, inclusive, and any individual value, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. The term "at least 80% identical" also includes any individual subranges therein, inclusive, such as "85% to 99%," "97% to 100%," and "90% to 100%." ​​The same applies to similar expressions, such as, but not limited to, "at least 70% identical," "at least 90% identical," and the like.

[0062] As used herein, the term "IC50" refers to the half-maximal inhibitory concentration (i.e., the concentration of a substance required for 50% inhibition in vitro). It is a measure of the potency or effectiveness of a substance in inhibiting a specific biological or biochemical function. IC50 values ​​are typically expressed as molar concentrations. The IC50 of an inhibitor can be determined by constructing a dose-response curve and examining the effect of different concentrations of the inhibitor on the specific biological or biochemical function in question.

[0063] As used herein, the term "inhibitory potency" refers to the effectiveness of a substance in inhibiting a specific biological or biochemical function, such as, but not limited to, the binding between a protein receptor and its ligand or the activation of a cellular receptor by its ligand. In some embodiments, inhibitory potency is determined by measuring the IC50 of the inhibitor for a specific ligand or substance. In this case, the relative inhibitory potency for different inhibitors and / or ligands can be assessed by comparing IC50 values. For example, a relative inhibitory potency of 3:1 means that the ratio of the IC50 values ​​of the two substances being compared is 3:1, where the first substance has a lower inhibitory potency (i.e., a higher IC50) than the second substance. A relative inhibitory potency of 1:3 means that the ratio of the IC50 values ​​of the two substances being compared is 1:3, where the first substance has a higher inhibitory potency (i.e., a lower IC50) than the second substance. Because the IC50 of an inhibitor can vary depending on the assay conditions, the relative inhibitory potency for different inhibitors and / or ligands is generally determined by comparing IC50 values ​​obtained under the same assay conditions. The terms "inhibition potency," "inhibitory potency," "inhibitory efficacy," and "neutralizing efficacy" are used interchangeably herein.

[0064] As used herein, the term "substantially the same" with respect to relative inhibitory potency means that two proteins have about the same relative inhibitory potency, e.g., no more than about 2-fold different (+ / - 2-fold), under the same experimental conditions, e.g., conditions where the ratio of the IC50 values ​​of the two proteins is about 2:1, 1:2, or 1:1.

[0065] As used herein, the term "functionally equivalent" refers to a variant sequence that has the same or substantially the same biological activity or function as the original sequence from which it is derived, e.g., a sequence that has no significant changes in physiological, chemical, physicochemical, or functional properties compared to the original sequence. The term "substantially identical" refers to a sequence that is functionally equivalent to an 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., salt and temperature conditions substantially equivalent to 0.5X SSC to about 5X SSC and 65°C for both hybridization and washing.

[0066] The term "dimer" refers to the presence of two polypeptides described herein in a TGFβ superfamily ligand binding agent (also referred to herein as a "binder"). A "homodimer" means that the two polypeptides have the same amino acid sequence, while a "heterodimer" means that the two polypeptides have different amino acid sequences.

[0067] The term "bivalent" refers to the presence of two TGFβR superfamily ligand binding regions (eg, ectodomains) in a TGFβ superfamily ligand binding agent.

[0068] As used herein, a "recombinant polypeptide" is a polypeptide made through the use of recombinant DNA technology or genetic engineering. In the context of this disclosure, a recombinant polypeptide is often referred to as a "polypeptide construct" or simply as a "polypeptide."

[0069] Proteins (including fragments thereof, preferably biologically active fragments, and peptides usually having fewer than 30 amino acids) comprise two or more amino acids coupled to each other via covalent peptide bonds (resulting in a chain of amino acids). As used herein, the term "polypeptide" describes a group of molecules usually consisting of more than 10 amino acids. The terms "polypeptide," "polypeptide chain," and "chain" are used interchangeably herein. Polypeptides can also form multimers, such as dimers, trimers, and higher oligomers, i.e., multimers consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are consequently referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally modified peptides / polypeptides / proteins, which are modified, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. As referred to herein, a "peptide," "polypeptide," or "protein" may also be chemically modified, such as by pegylation. Such modifications are well known in the art and are described herein.

[0070] As used herein, the terms "(specifically) bind," "(specifically) recognize," "specific for," "(specifically) directed," and "(specifically) react" mean that a polypeptide interacts or specifically interacts with a given target(s), such as a specific member(s) of the TGFβ superfamily of ligands. Specific binding is believed to be influenced by specific motifs in the amino acid sequence of the polypeptide. Binding is thus achieved as a result of their primary, secondary, and / or tertiary structure and secondary modifications of that structure. Specific interaction between a target-interaction site and its specific target may result in simple binding of the site to the target. Furthermore, specific interaction between a target-interaction site and its specific target may alternatively or additionally result in the initiation of a signal or may block the target from performing another activity, such as binding to an endogenous receptor, for example, due to induction of a conformational change in the target, oligomerization of the target, etc.

[0071] Generally, binding is considered specific when the binding affinity is about 10-12 to 10-9 M, 10-12 to 10-19 M, 10-11 to 10-9 M, or about 10-11 to 10-9 M. Whether a polypeptide or binding agent specifically reacts with or binds to a target can be readily tested, inter alia, by comparing the reaction of the polypeptide or binding agent with the target to the reaction of the polypeptide or binding agent with other proteins. In some embodiments, a polypeptide or binding agent of the present disclosure does not substantially bind to TGF-β superfamily ligands other than the desired ligand, e.g., does not substantially bind to BMP-9.

[0072] As used herein, the terms "does not substantially bind" or "cannot bind" mean that a polypeptide or binding agent of the disclosure exhibits no detectable binding to a given target, e.g., no more than 30%, no more than 20%, no more than 10%, or no more than 9%, 8%, 7%, 6%, 5%, or 3% reactivity with a given target.

[0073] As used herein, the term "selectively binds" refers to a polypeptide binding to a target site that is not shared with other proteins. Generally, a selective binding agent does not cross-react with other proteins and exclusively binds to a designated target protein(s). In the context of the present disclosure, "selective for activin A and GDF-8" means that a polypeptide or binding agent exclusively binds to or neutralizes activin A and GDF-8 ligands without substantially binding to or neutralizing other TGFβ superfamily ligands, such as BMP-9.

[0074] "Half-life" means the time it takes for 50% of an administered drug to be eliminated through biological processes, such as metabolism, excretion, etc.

[0075] "Hepatic first-pass metabolism" refers to the tendency of a drug to be metabolized upon first contact with the liver, i.e., during its first pass through the liver.

[0076] "Volume of distribution" refers to the degree of retention of a drug throughout the various compartments of the body, eg, intracellular and extracellular spaces, tissues and organs, and the distribution of the drug within these compartments.

[0077] "Extent of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins, such as albumin, resulting in a reduction or loss of the drug's biological activity.

[0078] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val), negatively charged side chains (e.g., Asp, Glu), positively charged side chains (e.g., Arg, His, Lys), or uncharged polar side chains (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0079] Similarly, "percent (%) nucleic acid sequence identity" with respect to a nucleic acid sequence of a polypeptide or binding agent identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the polypeptide or binding agent. A particular method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with the overlap span and overlap fraction set to 1 and 0.125, respectively.

[0080] TGFβ superfamily ligand binders In some embodiments, the present disclosure provides a TGFβ superfamily ligand binding agent comprising an ActRIIB-ECD region, a linker region, and an Fc domain (also referred to herein as a "binding agent" or "TGFβ ligand binding agent"). The individual components of the binding agents described herein are described in further detail in the following sections. In general, however, the binding agents described herein are dimeric proteins comprising two polypeptides, each comprising an ActRIIB-ECD, a peptide linker, and an Fc domain monomer. The two polypeptides assemble via the Fc domain monomer to form the dimeric binding agent described herein. See the schematic diagram in Figure 36. Upon assembly, 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, GDF-11, and BMP-10 and inhibit signaling of the one or more ligands through their respective receptors without substantially binding to BMP-9 and / or inhibiting BMP-9 signaling through its receptor. The binding agents may also have additional biological activities or functions, such as binding to other ligands or targets, as further described herein.

[0081] In some embodiments, the binding agents of the present disclosure comprise two polypeptide chains associated via an antibody Fc domain monomer, or via a constant CH2 domain, a constant CH3 domain, and / or a combination of CH2 and CH3. The antibody constant region can be derived from, or substantially identical to, a human IgG1, IgG2, IgG3, or IgG4 antibody. Association of both polypeptide chains generally occurs during protein expression and secretion, e.g., in mammalian cells. The Fc domain monomer is generally of human origin and typically comprises a CH2, CH3, or CH2 and CH3 from an antibody heavy chain, which provides disulfide bridges between the single polypeptide chains. In one embodiment, the Fc domain monomer provides at least one disulfide bond between the single polypeptide chains. In another embodiment, the Fc domain monomer provides at least two disulfide bonds between the single polypeptide chains. In some cases, the antibody heavy chain also provides for Protein A-based isolation of the dimeric polypeptide, e.g., after production in a host cell.

[0082] As noted above, certain TGFβ superfamily ligand binding agents and point mutations in the ECD have been described in the art. See, for example, WO2021 / 158675, WO2022 / 150590, WO2021 / 158675, WO2022 / 072882, WO2021 / 189019, and WO2021 / 189010. While point mutations in the ActRIIB ECD have been described in the context of other TGFβ superfamily ligand binding agents, the effects of these mutations in the context of these aforementioned agents do not predict the effects of these same mutations in the context of the binding agents described herein. Specifically, the data in the present application demonstrate that certain mutations in ActRIIB, when combined with peptide linkers of variable length, have unpredictable effects on TGFβ superfamily ligand binding.

[0083] For example, P121, P622, P624, P666, and P667 each have an F58E mutation in the ActRIIB-ECD. P121 and P624 have a 3-aa linker connecting the ActRIIB ECD and the Fc domain, P622 has a 14-aa linker connecting the ActRIIB ECD and the Fc domain, P666 has a 19-aa linker connecting the ActRIIB ECD and the Fc domain, and P667 has a 39-aa linker connecting the ActRIIB ECD and the Fc domain. As shown in Table 9 below, the combination of the F58E mutation with a longer linker (14 aa or longer) increased the inhibitory potency of the P622, P666, and P667 binders against activin A and activin B compared to the same mutation in combination with a short (3 aa) linker. The inhibitory potency against GDF-8 and GDF-11 remained similar between the short and long linker binders containing the F58E mutation (see Figures 10C and 10D). The F58E mutation combined with the longer linker (14 aa) also showed superior therapeutic efficacy in a rat model of PAH (see Example 5).

[0084] Similarly, the introduction of the D57E mutation into ActRIIB-ECD increased the inhibitory potency of the fusion protein when combined with a long linker (14 aa). For example, P759 has a 14 aa linker connecting the ActRIIB ECD and the Fc domain, and P120 has a 3 aa linker connecting the ActRIIB ECD and the Fc domain. P759 and P120 each contain a D57E mutation in ActRIIB-ECD. As shown in Table 9 and Figures 10E and 10F, P759 showed increased inhibitory potency for activin A, activin B, GDF-8, and GDF-11 (P120) compared to the same mutation combined with a short (3 aa) linker.

[0085] In contrast, the introduction of the K31Y or G27D mutation had completely different effects when combined with short and long linkers. The P124 and P761 binders each contain a K31Y mutation. The P758 and P119 binders each contain a G27D mutation. Combining either the K31Y or G27D mutation with a longer linker (14 aa or longer) reduced the inhibitory potency of the P124 and P758 binders against activin A, activin B, GDF-8, and GDF-11 (P119 and P761) compared with the same mutation combined with a short (3 aa) linker. See Figures 10G-10J.

[0086] Furthermore, the introduction of the V75Q mutation also showed unpredictable effects when combined with short and long linkers. The P762 and P126 binders each contained the V75Q mutation. However, in contrast to the F58E, K31Y, and G27D mutations described above, combining the V75Q mutation with different linker lengths did not affect the inhibitory potency against activin A, activin B, GDF-8, GDF-11, or BMP-9. The inhibitory potency of each ligand remained similar between the short and long linker agents containing the V75Q mutation. See Figures 10K and 10L.

[0087] Thus, the efficacy of a particular binding agent described herein is determined not only by the mutations contained in the extracellular ligand-binding domain, but also by the length of the linker used. As noted above, the combination of point mutations in the ActRIIB ECD and the length of the linker connecting the ActRIIB ECD to the Fc domain has unpredictable effects on binding to and inhibition of TGFβ superfamily ligands.

[0088] Additional ECD-based traps, such as luspatercept and sotatercept, are being clinically evaluated. Luspatercept (ACE-536, also known as REBLOZYL®) is a soluble fusion protein composed of a modified form of the extracellular domain of activin receptor type IIB (ActRIIB) linked to the Fc portion of human IgG1. Luspatercept inhibits several endogenous TGFβ superfamily ligands, thereby reducing Smad2 / 3 signaling. It is used to treat anemia in beta-thalassemia and myelodysplastic syndromes. For a description of luspatercept and other related fusion proteins, see, e.g., U.S. Patent Nos. 7,842,663, 8,058,229, 8,216,997, 8,252,900, 8,343,933, 8,361,957, 8,703,927, 9,138,459, 9,399,669, 9,439,945, 9,932,379, 10,131,700, 10,259,861, 10,689,427, and 10,829,532.

[0089] Sotatercept (also known as ACE-011) is a soluble decoy receptor composed of the extracellular domain of activin receptor type IIA (ActRIIA) linked to the Fc portion of human IgG1, capable of binding to and neutralizing activin and GDF. Sotatercept has been evaluated in healthy volunteers and patients with conditions characterized by dysfunctional TGF-β superfamily signaling, including hematologic disorders, bone loss, chemotherapy-induced anemia, multiple myeloma, myelodysplastic syndrome, β-thalassemia, and end-stage renal disease (Raftopoulos, H. et al., 2016; Abdulkadyrov, K. et al., 2014; Ruckle, J. et al., 2009; Komrokji, R. et al., 2018; Cappellini, MD et al., 2019; Coyne, D. et al., 2019; Sherman, M. et al., 2013). Recently, sotatercept has been evaluated for the treatment of pulmonary arterial hypertension (PAH).

[0090] By acting as a ligand trap for activin and GDF, sotatercept may correct the imbalance between the growth-promoting activin / growth differentiation factor pathway and the growth-inhibitory BMP pathway that occurs in PAH. In a phase 2 clinical trial in PAH patients, sotatercept was shown to reduce pulmonary vascular resistance (Humbert, M. et al., 2021). Additional PH clinical trials, including phase 3 trials, are ongoing or planned. For a description of sotatercept and other related fusion proteins, see, e.g., U.S. Patent Nos. 7,612,041, 7,709,605, 7,951,771, 7,988,973, 8,007,809, 8,629,109, 8,895,016, and 9,163,075. However, in several clinical studies of sotatercept, vascular and hematologic side effects have been found to be dose-limiting and limit potential therapeutic efficacy. For example, a multiple ascending dose study in healthy postmenopausal women planned to evaluate four doses—0.1, 0.3, 1 mg / kg, and 2 mg / kg—but was terminated early at the 1 mg / kg level because increases in hemoglobin, hematocrit, and red blood cell count were found to be dose-limiting (Sherman, M. Let et al., 2013). In a phase 2 clinical trial in patients with PAH, thrombocytopenia and increased hemoglobin levels were the most common hematologic adverse events, with 17% of patients receiving the 0.7 mg / kg dose experiencing increased hemoglobin (Humbert, M. et al., 2021). Such vascular and hematologic side effects are dose-limiting because they may not allow for the administration of the dose required for maximum efficacy, limiting the possibility of achieving maximum therapeutic benefit (Humbert, M. et al., 2021). In contrast, the binding agents provided herein (eg, P622 and P624) do not induce hematological effects in non-human primates, suggesting that these agents may have a broader therapeutic window than sotatercept.

[0091] In some embodiments, the binding agents of the present disclosure comprise homodimers, i.e., dimers of polypeptides having the sequence set forth in any one of SEQ ID NOs: 174-254, 333, or 339-341, or a sequence 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 other embodiments, the binding agents comprise heterodimers, i.e., dimers of two different polypeptides, at least one of which has the sequence set forth in any one of SEQ ID NOs: 174-254, 333, or 339-341, or a sequence 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.

[0092] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising ActRIIB-ECD comprising an F58E mutation and a long peptide linker. In some embodiments, the peptide linker is 10 amino acids or at least 10 amino acids in length. In some embodiments, the peptide linker is 14 amino acids or at least 14 amino acids in length. In some embodiments, the peptide linker is 19 amino acids or at least 19 amino acids in length. In some embodiments, the peptide linker is 39 amino acids or at least 39 amino acids in length. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 186, 190-194, 232-233, and 247-248, or a sequence 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.

[0093] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 186 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 190 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 191 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 192 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 193 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 194 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof.In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 232 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 233 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 247 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 248 or a sequence 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.

[0094] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising ActRIIB-ECD comprising an F58K mutation and a long peptide linker. In some embodiments, the peptide linker is 10 amino acids or at least 10 amino acids in length. In some embodiments, the peptide linker is 14 amino acids or at least 14 amino acids in length. In some embodiments, the peptide linker is 19 amino acids or at least 19 amino acids in length. In some embodiments, the peptide linker is 39 amino acids or at least 39 amino acids in length. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 213-216 or 242-244, or a sequence 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.

[0095] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 213, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 214, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 215, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 216, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 242, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 243, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof.In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 244 or a sequence 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.

[0096] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising ActRIIB-ECD comprising an F58Q mutation and a long peptide linker. In some embodiments, the peptide linker is 10 amino acids or at least 10 amino acids in length. In some embodiments, the peptide linker is 14 amino acids or at least 14 amino acids in length. In some embodiments, the peptide linker is 19 amino acids or at least 19 amino acids in length. In some embodiments, the peptide linker is 39 amino acids or at least 39 amino acids in length. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 220-223, 253, and 254, or a sequence 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.

[0097] In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 220, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 221, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 222, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 223, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 253, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or a functionally equivalent variant thereof. In some embodiments, a binding agent of the present disclosure comprises a dimer of a polypeptide comprising or consisting of SEQ ID NO: 254, or a sequence 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.

[0098] Activin receptor type IIB ectodomain variant As used herein, the term "activin receptor type IIB ectodomain variant" or "ActRIIB-ECD variant" refers to a polypeptide comprising the soluble extracellular portion of a single transmembrane receptor, ActRIIB, having at least one amino acid substitution with wild-type extracellular ActRIIB. The sequence of wild-type human ActRIIB-ECD is set forth in SEQ ID NO: 2 (Table 1). Unless otherwise specified, positions indicated for amino acid substitutions are numbered according to the amino acid sequence of SEQ ID NO: 2. For purposes of this disclosure, "human wild-type ActRIIB-ECD" refers to SEQ ID NO: 2.

[0099] In some embodiments, an ActRIIB-ECD variant comprises one or more amino acid substitutions at a position selected from G27, Q29, D30, K31, S38, D57, F58, V75, and F77. In some embodiments, an ActRIIB-ECD variant polypeptide comprises one or more amino acid substitutions selected from G27D, Q29Y, D30Q, K31Y, S38R, D57E, F58E, F58D, F58Y, F58K, F58Q, F58W, F58N, F58R, F58H, V75Q, and F77D. In some embodiments, an ActRIIB-ECD variant comprises one or more amino acid substitutions at a position selected from F58E, F58D, F58Y, F58K, F58Q, F58W, F58N, F58R, and F58H. In some embodiments, the ActRIIB-ECD variant comprises one or more amino acid substitutions selected from F58E, F58K, and F58Q. In some embodiments, the ActRIIB ECD variant comprises an amino acid substitution of F58E. In some embodiments, the ActRIIB ECD variant comprises an amino acid substitution of F58K. In some embodiments, the ActRIIB ECD variant comprises an amino acid substitution of F58Q. Other amino acid substitutions in ActRIIB-ECD are known in the art (e.g., WO2021 / 158675, WO2022 / 150590, WO2021 / 158675, WO2022 / 072882, WO2021 / 189019, and WO2021 / 189010, each of which is incorporated herein by reference). These additional mutations, in combination with the linkers described herein, can be incorporated into the binding agents described herein to alter the ligand binding properties of ActRIIB-ECD.

[0100] In some embodiments, the ActRIIB-ECD variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-22, 331, 332, or 24-33. In some embodiments, the ActRIIB-ECD variant comprises at least 85% (e.g., at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more) amino acid sequence identity to the sequence of wild-type human ActRIIB-ECD. In some embodiments, the ActRIIB-ECD variant may have at least 85% (e.g., at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more) amino acid sequence identity to the sequence set forth in SEQ ID NO: 2.

[0101] In some embodiments, the amino acid sequence of the ActRIIB-ECD variant comprises at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 4-22, 331, 332, or 24-33. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position F58 and comprises an amino acid sequence that is 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: 5-13. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position F58 and comprises or consists of an amino acid sequence selected from SEQ ID NOs: 5-13. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position F58 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 F58 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 F58 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 F58 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 F58 and comprises or consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position F58 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 F58 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 F58 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 F58 and comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0102] In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position D57 and comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to SEQ ID NO: 4. In some embodiments, the ActRIIB-ECD variant comprises an amino acid substitution at position D57 and comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0103] An exemplary ActRIIB ECD is provided in Table 1. Amino acid substitutions are shown in bold and expanded text. [Table 1-1] [Table 1-2]

[0104] In some embodiments, an ActRIIB-ECD variant of the disclosure further comprises an extension of up to five amino acids at the N-terminus. In some embodiments, an ActRIIB-ECD variant of the disclosure further comprises a five amino acid extension at the N-terminus, e.g., GRGEA (SEQ ID NO: 23). In some embodiments, an ActRIIB-ECD variant of the disclosure further comprises an extension at the N-terminus of four, three, two, or one amino acid, e.g., but not limited to, RGEA, GEA, EA, or A. Exemplary ActRIIB ECDs with N-terminal extensions are provided in Table 2. N-terminal extension amino acids are shown in bold and italic text. Amino acid substitutions are shown in bold and expanded text. [Table 2]

[0105] In some embodiments, the ActRIIB-ECD variants of the present disclosure further comprise an alanine-proline-threonine (APT) extension at the C-terminus. As shown in Figures 10O-10P, the addition of the C-terminal extension APT to ActRIIB-ECD does not affect inhibitory potency. Thus, in some embodiments, any one of SEQ ID NOs: 2-22, 331, 332, and 24-33 can further comprise an APT extension at the C-terminus.

[0106] The ActRIIB-ECD variants of the present disclosure are designed to maximize therapeutic efficacy in a particular disease indication while minimizing adverse effects, specifically, to prevent or reduce inhibition of endogenous BMP-9 signaling while maintaining and / or increasing neutralizing potency against other TGFβ superfamily ligands, such as activin A, activin B, GDF-8, GDF-11, and / or BMP-10. The ActRIIB-ECD variants of the present disclosure exhibit: (1) similar or improved binding to activin A, activin B, GDF-8, GDF-11, and / or BMP-10 compared to wild-type ActRIIB-ECD (allowing them to compete with endogenous receptors for ligand binding and reduce or inhibit endogenous receptor signaling), and (2) reduced or eliminated binding to BMP-9 compared to wild-type ActRIIB-ECD (allowing them to avoid toxicity associated with inhibition of BMP-9 signaling). These variants can be used to treat a wide range of diseases and conditions in which activin receptor signaling is elevated, such as pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or hybrid PH), metabolic 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 damage), muscle diseases, fibrosis, and low red blood cell levels (e.g., anemia, blood loss), as further described herein. The variants may result in, for example, but not limited to, a reduction in the symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or hybrid PH), a reduction in bone resorption or osteoclast activity, an increase in bone formation or bone mineral density, an increase in muscle mass or strength, a reduction in fibrosis (e.g., a decrease in fibrosis or a slowing or halting of the progression of fibrosis), and / or an increase in red blood cell levels (e.g., an increase in hemoglobin levels, hematocrit, or red blood cell count, e.g., an increase in red blood cell production), as further described herein.

[0107] In some embodiments, the ActRIIB-ECD variants of the present disclosure bind to one or more ligands selected from activin A, activin B, GDF-8, GDF-11, and BMP-10, and inhibit signaling through the respective receptors of the one or more ligands without substantially binding to BMP-9 and / or inhibiting BMP-9 signaling through its receptor.

[0108] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against human BMP-9 signaling is reduced by about 100-fold compared to the inhibitory potency of human wild-type ActRIIB-ECD against human BMP-9 signaling.

[0109] In some embodiments, the inhibitory potency of the ActRIIB-ECD variants of the present disclosure against one or more ligands selected from activin A, activin B, GDF-8, GDF-11, and BMP-10 is increased compared to or substantially the same as the inhibitory potency of human wild-type ActRIIB-ECD against the same one or more ligands.

[0110] 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 compared to human wild-type ActRIIB-ECD.

[0111] In some embodiments, the ActRIIB-ECD variants of the present disclosure do not cause vascular complications in a subject.

[0112] In some embodiments, the ActRIIB-ECD variants of the present disclosure do not increase vascular permeability or leakage in a subject.

[0113] As a result, according to the present disclosure, novel polypeptides are provided herein comprising activin receptor type IIB (ActRIIB) ectodomain (ECD) variants, which have one or more amino acid substitutions compared to the sequence of human wild-type ActRIIB-ECD and have tailored TGFβ superfamily ligand specificity aimed at preventing or reducing disruption of endogenous BMP-9 signaling while maintaining and / or increasing the neutralization potency of other TGFβ superfamily ligands, such as activin A, activin B, GDF-8, GDF-11, and / or BMP-10, in order to maximize therapeutic efficacy while minimizing adverse effects.

[0114] Polypeptides containing ActRIIB ECD variants In some embodiments, the present disclosure provides a polypeptide comprising an ActRIIB ECD variant fused to an Fc domain monomer via a linker. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, an ActRIIB ECD variant-peptide linker-Fc domain monomer. The polypeptide comprising the ActRIIB ECD can dimerize via cysteine ​​bonds between the Fc domain monomers to form a TGFβ superfamily ligand binding agent described herein.

[0115] Linker In some embodiments, the ActRIIB ECD variant described herein is fused to a heterologous domain via a linker. In some embodiments, the heterologous domain increases the stability of the polypeptide. In some embodiments, the heterologous domain is selected from the group consisting of an Fc domain monomer (e.g., a wild-type Fc domain monomer, an Fc domain monomer with one or more amino acid substitutions), an albumin-binding peptide, a fibronectin domain, or a human serum albumin domain.

[0116] As used herein, the terms "peptide linker" and "linker" are used interchangeably to refer to a short stretch of amino acids used to connect two functional domains together within a polypeptide chain. For example, in some embodiments of the polypeptides or binding agents of the present disclosure, the ActRIIB-ECD variant and the Fc domain monomer are linked together on the polypeptide chain via one or more peptide linkers. Peptide linkers can also be used to attach other domains, modules, or regions (such as half-life extending domains) to the polypeptides or binding agents of the present disclosure. As used herein, the term "long linker" refers to a linker that is at least 10 amino acids long (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids long). As used herein, the term "short linker" refers to a linker that is less than 10 amino acids long (i.e., 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long).

[0117] 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 may contain a GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 92), GGGS (SEQ ID NO: 91), GGGG (SEQ ID NO: 69), GGGGA (SEQ ID NO: 90), GGGGS (SEQ ID NO: 68), GGGGG (SEQ ID NO: 89), GGAG (SEQ ID NO: 88), GGSG (SEQ ID NO: 87), AGGG (SEQ ID NO: 86), or SGGG (SEQ ID NO: 76) motif, e.g., multiple or repeated motifs.

[0118] In some embodiments, the linker can contain 2 to 12 amino acids including a GA or GS motif, e.g., GA, GS, GAGA (SEQ ID NO: 103), GSGS (SEQ ID NO: 95), GAGAGA (SEQ ID NO: 96), GSGSGS (SEQ ID NO: 97), GAGAGAGA (SEQ ID NO: 98), GSGSGSGS (SEQ ID NO: 99), GAGAGAGAGA (SEQ ID NO: 100), GSGSGSGSGS (SEQ ID NO: 101), GAGAGAGAGAGA (SEQ ID NO: 102), and GSGSGSGSGSGSGS (SEQ ID NO: 104). In some embodiments, the linker can contain 3 to 12 amino acids including a GGA or GGS motif, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 105), GGSGGS (SEQ ID NO: 106), GGAGGAGGA (SEQ ID NO: 107), GGSGGSGGS (SEQ ID NO: 108), GGAGGAGGAGGA (SEQ ID NO: 109), and GGSGGSGGSGGS (SEQ ID NO: 110). In some embodiments, the linker can contain 4 to 12 amino acids, including the motifs GGAG (SEQ ID NO: 111), GGSG (SEQ ID NO: 112), GGAGGGAG (SEQ ID NO: 113), GGSGGGSG (SEQ ID NO: 114), GGAGGGAGGGAG (SEQ ID NO: 115), and GGSGGGSGGGSG (SEQ ID NO: 116). In some embodiments, the linker can contain the motifs GGGGA (SEQ ID NO: 90) or GGGGS (SEQ ID NO: 68), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 117) and GGGGSGGGGSGGGGGS (SEQ ID NO: 58). In some embodiments, the amino acid linker between the ActRIIB-ECD variant and the heterologous domain (e.g., an Fc domain monomer (e.g., a wild-type Fc domain monomer, an Fc domain monomer with one or more amino acid substitutions), an albumin-binding peptide, a fibronectin domain, or a human serum albumin domain) can be GGG, GGGA (SEQ ID NO: 92), GGGG (SEQ ID NO: 69), GGGAG (SEQ ID NO: 335), GGGAGG (SEQ ID NO: 336), or GGGAGGG (SEQ ID NO: 337).

[0119] When a linker is used, the linker is generally of sufficient length and sequence to ensure that each of the domains can retain their differential binding specificity and / or function independently of one another. In some embodiments, a peptide linker is selected that does not further promote any secondary structure. The linkage of the domains to one another can be provided by genetic engineering, for example, as described herein. Methods for preparing fused and operably linked polypeptide constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0120] In some embodiments, the linker comprises various permutations of amino acid sequences containing Gly and Ser. In some embodiments, the linker is a glycine- and serine-rich linker. In some embodiments, the linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, or 2-3 glycine residues) or glycine and proline residues, and may contain, for example, a single threonine / serine and glycine sequence, a repeating sequence of threonine / serine and / or glycine, e.g., a single or repeating sequence of GGG, GGGG (SEQ ID NO: 69), GGGS (SEQ ID NO: 91), TGGGG (SEQ ID NO: 74), SGGGG (SEQ ID NO: 75), TGGG (SEQ ID NO: 73), or SGGG (SEQ ID NO: 76). Other near-neutral amino acids, such as, but not limited to, Thr, Asn, Pro, and Ala, may also be used in the linker sequence.

[0121] In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is more than 10 amino acids in length. In some embodiments, the linker is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, the linker is less than 40, 35, 30, 25, 22, or 20 amino acids. In some embodiments, the linker is 10 to 50, 10 to 40, 10 to 30, 10 to 25, 10 to 21, 10 to 15, 10 to 14, 12 to 14, 15 to 25, 17 to 22, 20, or 21 amino acids in length. In some embodiments, the linker is 14 to 40, 14 to 39, 14 to 35, 14 to 30, 14 to 25, or 14 to 20 amino acids in length. In some embodiments, the linker is at least 10 amino acids in length. In some embodiments, the linker is at least 14 amino acids in length. In some embodiments, the linker is at least 19 amino acids in length. In some embodiments, the linker is at least 39 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 39 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In further embodiments, the linker has a length of at least 12, 14, 15, 20, 21, 25, 30, 35, 40, 45, or 50 amino acids.

[0122] In some embodiments, the linker comprises SEQ ID NO: 59. In some embodiments, the linker comprises SEQ ID NO: 54. In some embodiments, the linker comprises SEQ ID NO: 34. In some embodiments, the linker comprises SEQ ID NO: 63.

[0123] In some embodiments, the linker consists of SEQ ID NO: 59. In some embodiments, the linker consists of SEQ ID NO: 54. In some embodiments, the linker consists of SEQ ID NO: 34. In some embodiments, the linker consists of SEQ ID NO: 63.

[0124] In some embodiments, the linker comprises or consists of a sequence set forth in any one of SEQ ID NOs: 34-133 and 335-337.

[0125] In some embodiments, the linker is a glycine-rich, often glycine / serine-rich, peptide of up to 40 amino acids, or 1-40 amino acids, 2-39 amino acids, 3-39 amino acids, 3-14 amino acids, 3-19 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, or 15-25 amino acids. In some embodiments, the peptide linker contains a relatively small number of amino acid residues, e.g., 39 or fewer amino acids, 19 or fewer amino acids, 14 or fewer amino acids, 5 or fewer amino acids, or 3 or fewer amino acids. In certain embodiments, Gly-rich linkers are used. In one embodiment, the peptide linker can consist of the single amino acid glycine (Gly). In another embodiment, the peptide linker comprises or consists of the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser, or a polymer thereof, i.e., (Gly4Ser)n, where n is an integer of 1 or greater, or n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0126] In some embodiments, the linker comprises the amino acid sequence GlyGlyGlyGlySer(GGGGS) (SEQ ID NO: 68), or repeats thereof (GGGGS)n, where n>2. In certain embodiments, n>3, or n=3-10. In some embodiments, n>4, or n=4-10. In some embodiments, n is 4 or less in a (GGGGS)n linker. In some embodiments, n=4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6. In some embodiments, n=3, 4, 5, 6, or 7. In some embodiments, n=4. In some embodiments, linkers comprising a (GGGGS)n sequence also include an N-terminal threonine.

[0127] In some embodiments, the linker may contain amino acids other than glycine, alanine, and serine, for example, AAAL (SEQ ID NO: 118), AAAK (SEQ ID NO: 119), AAAR (SEQ ID NO: 120), EGKSSGSGSESKST (SEQ ID NO: 121), GSAGSAAGSGEF (SEQ ID NO: 122), AEAAAKEAAAKA (SEQ ID NO: 123), KESGSVSSEQLAQFRSLD (SEQ ID NO: 124), GENLYFQSGG (SEQ ID NO: 125), SACYCELS (SEQ ID NO: 126), RSIAT (SEQ ID NO: 127), RPACKIPNDLKQKVMNH (SEQ ID NO: 128), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 129), AAANSSIDLISVPVDSR (SEQ ID NO: 130), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 131). In some embodiments, the linker can contain a motif, e.g., multiple or repeated motifs, of EAAAK (SEQ ID NO: 132). In some embodiments, the linker can contain a motif, e.g., multiple or repeated motifs, of a praline-rich sequence 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: 133).

[0128] The length of the peptide linker and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the linker can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0129] Non-limiting examples of linkers are shown in Table 3. It should be understood that the linker is not intended to be particularly limited and any suitable linker may be used, so long as the desired function of the polypeptide or binding agent (binding, neutralization, etc.) is provided. [Table 3-1] [Table 3-2] [Table 3-3]

[0130] In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises one or more linkers having a sequence set forth in any one of SEQ ID NOs: 59, 54, 34, or 63. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent comprises a glycine-rich linker 2, 3, 6, 10, 14, 19, or 39 amino acids in length at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises a linker of SEQ ID NO: 59 at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises a linker of SEQ ID NO: 54 at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the present disclosure comprises a linker of SEQ ID NO: 34 at the C-terminus of the ActRIIB ECD variant polypeptide. In some embodiments, an ActRIIB ECD variant polypeptide or binding agent of the disclosure comprises a linker of SEQ ID NO: 63 at the C-terminus of the ActRIIB ECD variant polypeptide.

[0131] Fc domain monomers and Fc domains In some embodiments, the present disclosure provides a polypeptide comprising an ActRIIB-ECD variant described herein fused to an Fc domain monomer via a linker, hi some embodiments, the ActRIIB-ECD variant is fused C-terminally to the N-terminus of the Fc domain monomer via a linker.

[0132] As used herein, an "Fc domain monomer" describes a single-chain protein that, when associated with another Fc domain monomer, forms a functional Fc domain. The association of two Fc domain monomers creates one Fc domain. As used herein, an "Fc domain" describes the minimum region (in the context of a larger polypeptide) or the minimum protein fold (in the context of an isolated protein) that can bind to or be bound by an Fc receptor (FcR). When two Fc domain monomers associate, the resulting Fc domain has Fc receptor binding activity. Thus, the Fc domain is a dimeric structure that can bind to an Fc receptor. Unless otherwise specified, all references herein to a "variant Fc domain" should be understood to refer to a dimeric Fc domain, where each Fc domain monomer contains the referenced mutation.

[0133] As used herein, an Fc domain is understood to include a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains (CH2, CH3) of an IgG, and optionally, the flexible hinge N-terminal to these domains. Although the boundaries of an Fc domain monomer may vary, a human IgG heavy chain Fc domain monomer is usually defined as including residues C226 or P230 at its carboxy terminus. Unless otherwise specified, all references to amino acid positions in Fc domains and Fc domain monomers follow the EU index as set forth in Kabat (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). Fc may refer to this region in isolation or in the context of a polypeptide construct. It should be noted that polymorphism has been observed at many Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist between the sequences provided herein and those in the art. The Fc domain monomers comprised in the polypeptides or binding agents of the present disclosure may be IgG1, IgG2, IgG3, or IgG4 domains.

[0134] In exemplary embodiments, the polypeptides of the present disclosure comprise one or more constant regions of an antibody, e.g., the second constant domain (CH2) and / or the third constant domain (CH3) of an antibody heavy chain, or an Fc domain monomer of an antibody heavy chain. The antibody may be, for example, an IgG antibody, such as, but not limited to, an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the antibody is a human antibody, e.g., the Fc domain monomer comprises the constant region of a human IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the Fc domain monomer has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a human IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the Fc domain monomer comprises or consists of an Fc domain monomer of a human IgG1 antibody. In another specific embodiment, the Fc domain monomer comprises or consists of an Fc domain monomer of a human IgG2 antibody. In another specific embodiment, the Fc domain monomer comprises or consists of an Fc domain monomer of a human IgG4 antibody. Exemplary Fc domain sequences (including both the wild-type sequence, its polymorphs, and variant sequences) are provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0135] Generally, the ActRIIB-ECD polypeptide is configured such that the Fc domain monomer is linked at its N-terminus to the C-terminus of the ActRIIB-ECD variant, and the orientation of the construct for each ActRIIB-ECD polypeptide is a single chain of (ActRIIB-ECD variant)-(linker)-(Fc domain monomer) from N-terminus to C-terminus. However, the orientation of the construct is not particularly limited, and other orientations are contemplated. For example, in some embodiments, the Fc domain monomer may be linked at its C-terminus to the N-terminus of the ActRIIB-ECD variant.

[0136] In exemplary embodiments, an Fc domain monomer allows for the covalent assembly of two or more polypeptide chains, e.g., via disulfide bonds between cysteine ​​residues. In this manner, the Fc domain monomer acts as a dimerization domain, allowing the assembly of two ActRIIB-ECD polypeptide chains to form a dimer. According to the present disclosure, such dimers generally comprise two polypeptides, each comprising an ActIIRB-ECD variant linked to an Fc domain monomer described herein, thereby forming a bivalent TGFβ superfamily ligand binder. Thus, the binders described herein comprise two ActIIRB-ECD variants, a linker domain, and an Fc domain.

[0137] An Fc domain monomer generally contains one or more cysteine ​​residues for cross-linking a first polypeptide with a second polypeptide in a homodimeric construct. For example, an Fc domain monomer may contain at least two cysteine ​​residues for forming disulfide bridges between two polypeptides, thereby forming a dimer. In some embodiments of the present technology, an Fc domain monomer comprises or consists of a sequence set forth in any one of SEQ ID NOs: 134-173 and 338, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, an Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 134. In certain embodiments, an Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135. In certain embodiments, an Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 137. In certain embodiments, the Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 138. In certain embodiments, the Fc domain monomer comprises or consists of the amino acid sequence set forth in SEQ ID NO: 157.

[0138] In some embodiments, the present disclosure provides binding agents comprising a variant Fc domain, i.e., a non-naturally occurring Fc domain, e.g., an Fc domain that includes one or more non-naturally occurring amino acid residues, substitutions, additions, deletions, etc.

[0139] In some embodiments of the present technology, the Fc domain is a variant Fc domain that forms a variant Fc domain on a polypeptide or binding agent that has desirable properties, such as increased half-life, compared to a naturally occurring (wild-type) Fc sequence. As used herein, "variant Fc domain" refers to a non-naturally occurring Fc domain, e.g., an Fc domain that includes one or more non-naturally occurring amino acid residues, one or more amino acid substitutions relative to a wild-type human constant domain, or one or more amino acid deletions, additions, and / or modifications.

[0140] There are many known polymorphs for the IgG1 Fc domain, including the "DEL" polymorph and the "EEM" polymorph. The DEL polymorph contains the amino acids DEL at positions 356, 357, and 358, respectively (also referred to herein as "Fc-DL," e.g., SEQ ID NO: 135). The EEM polymorph contains the amino acids EEM at positions 356, 357, and 358, respectively (also referred to herein as "Fc-EM," e.g., SEQ ID NO: 134). Two binding agents that are otherwise identical except for the presence of a DEL or EEM Fc domain are expected to exhibit similar properties in terms of ligand binding and therapeutic efficacy. In some embodiments of the present technology, the Fc domain is a DEL Fc domain ("DL"). In some embodiments of the present technology, the Fc domain is an EEM Fc domain ("EM"). Other polymorphs may also be used, for example, the IgG1 polymorphs of SEQ ID NOs: 134 to 135 and 139 to 147, the IgG2 polymorphs of SEQ ID NOs: 148 to 157, the IgG3 polymorphs of SEQ ID NOs: 158 to 164, and the IgG4 polymorphs of SEQ ID NOs: 165 to 173.

[0141] In some embodiments, a variant Fc domain formed by two variant Fc domain monomers has altered binding characteristics to an Fc receptor, such as FcRn, compared to a comparable molecule (e.g., a protein having the same amino acid sequence except for having a wild-type Fc domain monomer). The serum half-life of a protein comprising an Fc domain can be increased by increasing the binding affinity of the Fc domain for FcRn. In one embodiment, the Fc domain variant has an enhanced serum half-life relative to a comparable molecule. In certain embodiments, the Fc domain variant comprises at least one amino acid substitution at one or more positions selected from the group consisting of M252Y, S254T, and T256 (referred to herein as "YTE"; e.g., SEQ ID NO: 137). In another embodiment, the Fc domain variant comprises a Y at position 252 (e.g., SEQ ID NO: 138, referred to herein as "Fc-Y"). In another embodiment, the Fc domain variant comprises a T at position 254. In another embodiment, the Fc domain variant comprises an E at position 256.

[0142] Thus, in some embodiments of the present technology, an ActRIIB-ECD polypeptide comprises a variant Fc domain monomer that forms an Fc domain with increased in vivo half-life relative to a comparable molecule, hi some such embodiments, the Fc domain monomer of the ActRIIB-ECD polypeptide comprises a substitution of at least one amino acid residue selected from the group consisting of residues 252, 254, and 256.

[0143] In some embodiments, the ActRIIB-ECD polypeptide comprises a variant Fc domain monomer comprising at least one amino acid substitution selected from the group consisting of M252Y, S254T, and T256E. In such embodiments, the variant Fc domain monomer may further comprise one or more additional amino acid substitution(s), such as, but not limited to, E356D and M358L.

[0144] In some embodiments, the ActRIIB-ECD polypeptide comprises a variant Fc domain monomer comprising the following amino acid substitutions: M252Y, S254T, and T256E (referred to herein as "FcYTE" or "YTE"). In some embodiments, the FcYTE domain monomer is the DEL polymorph (referred to herein as YTE-DL, e.g., SEQ ID NO: 137). In some embodiments, the FcYTE domain monomer is the EEM polymorph (referred to herein as YTE-EM, e.g., SEQ ID NO: 136).

[0145] In some embodiments, the ActRIIB-ECD polypeptide comprises a variant Fc domain monomer comprising the following amino acid substitution: M252Y (referred to herein as "FcY"). In some embodiments, the FcY domain monomer is in the DEL polymorph (referred to herein as Y-DL, e.g., SEQ ID NO: 138). In some embodiments, the FcY domain monomer is in the EEM polymorph (referred to herein as Y-EM, e.g., SEQ ID NO: 338).

[0146] In some embodiments, the ActRIIB-ECD polypeptide comprises an Fc domain monomer comprising a lysine residue (K) at the C-terminus.

[0147] In some embodiments, a variant Fc domain for use in an ActRIIB-ECD polypeptide of the present disclosure (e.g., an Fc domain formed by two variant Fc domain monomers) comprises one or more amino acid substitutions that reduce aggregation and / or increase stability and / or half-life of the ActRIIB-ECD polypeptide compared to a naturally occurring Fc sequence. In some embodiments, the Fc domain is selected to provide one or more effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation (complement-dependent cytotoxicity or CDC), opsonization, etc. In one embodiment, the variant Fc domain has enhanced binding to an Fc receptor compared to a comparable molecule. In a specific embodiment, the variant Fc domain has enhanced binding to the neonatal Fc receptor FcRn. In another embodiment, the variant Fc domain and / or polypeptide or binding agent containing the variant Fc domain has a binding affinity for FcRn that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold greater than that of a comparable molecule. The serum half-life of a protein comprising an Fc domain can be increased by increasing the binding affinity of the Fc domain monomer for FcRn. Thus, in one embodiment, a polypeptide or binding agent comprising a variant Fc domain has an enhanced serum half-life compared to a comparable molecule.

[0148] Examples of means for extending the serum half-life of the polypeptides and binding agents of the present disclosure include peptides, proteins, or protein domains fused to or otherwise attached to the polypeptides and binding agents. The group of peptides, proteins, or protein domains includes peptides that bind to other proteins with favorable pharmacokinetic profiles in the human body, such as serum albumin (see WO 2009 / 127691). An alternative concept for such half-life extending peptides includes peptides that bind to the neonatal Fc receptor (FcRn, see WO 2007 / 098420), which can also be used in the polypeptides and binding agents of the present disclosure. The concept of attaching larger domains of proteins or complete proteins includes, for example, fusions of human serum albumin, variants or mutants of human serum albumin (WO2011 / 051489, WO2012 / 059486, WO2012 / 150319, WO2013 / 135896, WO2014 / 072481, WO2013 / 075066) or domains thereof, as well as fusions of immunoglobulin constant regions (Fc domains) and variants thereof, as described herein. Such variants of the Fc domain may be optimized / modified to enable desired dimer or multimer pairing, to eliminate Fc receptor binding (e.g., Fcg receptors), to enhance binding to FcRn, or for other reasons. Another concept known in the art for extending the half-life of small protein compounds in the human body is PEGylation of such compounds, such as the polypeptides or binding agents of the present disclosure.

[0149] In one embodiment, the disclosure provides a binding agent, wherein the Fc domain is 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, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 347, 251, 252, 254, 255, 256, 262 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. Optionally, the Fc domain may comprise non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, 6,737,056, PCT Patent Publication Nos. WO 01 / 58957, WO 02 / 06919, WO 04 / 06920, and WO 05 / 06921). 16750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925, and WO06 / 020114). In certain embodiments, the present disclosure provides Fc variant protein compositions, wherein the Fc domain is 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, as numbered by the EU index as set forth in Kabat. , 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,2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265 Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 26 9R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 2 97D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328 E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 33 0Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 434W. Optionally, the Fc domain may include additional and / or alternative amino acid substitutions known to those skilled in the art (see, e.g., U.S. Patent Nos. 5,624,821, 6,277,375, 6,737,056, PCT Patent Publication Nos. WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, and WO05 / 040217).

[0150] Additional Domains It is envisioned that the ActRIIB-ECD polypeptides and / or binding agents of the present disclosure may have additional binding specificities or additional functions in addition to their function of binding to the target TGFβ superfamily ligand(s) as specified. In some embodiments of the present technology, the ActRIIB-ECD polypeptides or binding agents may be conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, and / or a diagnostic moiety. In some embodiments, the polypeptides may have additional functions, such as a fully functional Fc constant domain that mediates antibody-dependent cellular cytotoxicity, such as through the recruitment of effector cells such as NK cells, by providing a label (e.g., fluorescent), by providing a therapeutic agent such as a toxin or radionuclide, and / or by providing a means to enhance serum half-life.

[0151] In some embodiments, the ActRIIB-ECD polypeptide described herein comprises ActRIIB-ECD, a linker, an Fc domain monomer, and one or more additional domains. In some embodiments, the one or more additional domains are selected from a fibronectin domain and a human serum albumin domain. As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to membrane-spanning receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID No. P02751. In other embodiments, the fibronectin domain is an adnectin protein.

[0152] In some embodiments, the polypeptide or binding agent of the present disclosure comprises an ActRIIB-ECD variant fused to one or more fibronectin domains. Binding to a fibronectin domain can improve the pharmacokinetics of a protein pharmaceutical. A fibronectin domain is a high-molecular-weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to membrane-spanning receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments of the present invention, a fibronectin domain is conjugated to the N-terminus or C-terminus (e.g., the C-terminus) of an ActRIIB-ECD variant described herein (e.g., an ActRIIB-ECD variant having an amino acid sequence set forth in any one of SEQ ID NOS: 4-22, 331-332, and 24-33), thereby increasing the serum half-life of the ActRIIB-ECD variant. The fibronectin domain can be conjugated directly or through a linker to the N-terminus or C-terminus of an ActRIIB-ECD variant, or the polypeptide, or the binding agent. In some embodiments, the polypeptide or binding agent of the present disclosure can be fused to the N- or C-terminus of a fibronectin domain, for example, through conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the ActRIIB-ECD variant and the fibronectin domain. Without being bound by theory, it is expected that in some embodiments, the inclusion of a fibronectin domain in the ActRIIB-ECD variants described herein can result in long-term retention of the therapeutic protein through binding to integrins and extracellular matrix components such as collagen and fibrin.

[0153] By way of example, fibronectin domains that can be used in the methods, compositions, and polypeptides of the present disclosure are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID No. P02751. In another embodiment, the fibronectin domain is an Adnectin protein.

[0154] 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 the blood. It constitutes approximately half of the serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID number P02768.

[0155] 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 pharmaceuticals. Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and constitutes approximately half of serum proteins. It is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure necessary for proper distribution of body fluids between blood vessels and body tissues. In some embodiments, the serum albumin is human serum albumin. In some embodiments, human serum albumin is conjugated to the N-terminus or C-terminus (e.g., the C-terminus) of an ActRIIB-ECD variant described herein (e.g., an ActRIIB-ECD variant having an amino acid sequence set forth in any one of SEQ ID NOS: 4-22, 331-332, and 24-33) to increase the serum half-life of the ActRIIB-ECD variant. Human serum albumin can be conjugated to the N-terminus or C-terminus of the ActRIIB-ECD variant directly or through a linker.

[0156] By way of example, serum albumin that can be used in the polypeptides and methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID number P02768. In some embodiments, the polypeptide or binding agent of the present disclosure can be fused to the N- or C-terminus of human serum albumin, for example, through conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the ActRIIB-ECD variant and human serum albumin. Without being bound by theory, it is expected that in some embodiments, the inclusion of human serum albumin in the ActRIIB-ECD variants described herein can result in long-term retention of the therapeutic protein.

[0157] In some embodiments, a polypeptide or binding agent of the present disclosure further comprises a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization, an albumin-binding peptide, a fibronectin domain, or human serum albumin), which may be fused to the N-terminus or C-terminus (e.g., the C-terminus) of an ActRIIB-ECD variant, polypeptide, or binding agent via a linker or other covalent bond. A polypeptide comprising an ActRIIB-ECD variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or heterodimer) through interaction between two Fc domain monomers, which combine to form the Fc domain in the dimer. Further, in some embodiments, a polypeptide or binding agent described herein has a serum half-life of at least 7 days in humans.

[0158] Exemplary TGFβ Superfamily Binding Agents 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. [Table 5-1] Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21

[0159] In some embodiments, the binding agent comprises, from N-terminus to C-terminus, an ActRIIB ECD, a peptide linker, and an Fc domain. In some embodiments, the ActRIIB ECD comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions are at a position selected from G27, Q29, D30, K31, S38, D57, F58, V75, and F77, where amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the amino acid substitution at position G27 is G27D. In some embodiments, the amino acid substitution at position Q29 is Q29Y. In some embodiments, the amino acid substitution at position D30 is D30Q. In some embodiments, the amino acid substitution at position K31 is K31Y. In some embodiments, the amino acid substitution at position S38 is S38R. In some embodiments, the amino acid substitution at position D57 is D57E. In some embodiments, the amino acid substitution at position F58 is selected from F58D, F58E, F58K, F58Q, F58W, F58N, F58R, F58H, and F58Y. In some embodiments, the amino acid substitution at position F58 is F58E. In some embodiments, the amino acid substitution at position F58 is F58K. In some embodiments, the amino acid substitution at position F58 is F58Q. In some embodiments, the amino acid substitution at position V75 is V75Q. In some embodiments, the amino acid substitution at position F77 is F77D.

[0160] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a G27D amino acid substitution, a peptide linker that is 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 that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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 (P119). 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 (P119).

[0161] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a D57E amino acid substitution, a peptide linker that is 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 that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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 (P120). 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 (P120).

[0162] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 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:5, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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:5, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 (P121). 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 (P121).

[0163] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a Q29Y amino acid substitution, a peptide linker that is 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: 14, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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: 14, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 (P122). 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 (P122).

[0164] In some embodiments, the TGFβ superfamily ligand-binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a D30Q amino acid substitution, a peptide linker that is 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: 15, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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 (P123). 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 (P123).

[0165] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a K31Y amino acid substitution, a peptide linker that is 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: 16, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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 (P124). 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 (P124).

[0166] In some embodiments, the 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 S38R amino acid substitution, a peptide linker that is 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: 17, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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 (P125). 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 (P125).

[0167] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a V75Q amino acid substitution, a peptide linker that is 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: 18, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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: 184 (P126). 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 (P126).

[0168] In some embodiments, the 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 F77D amino acid substitution, a peptide linker that is 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 the amino acid sequence of SEQ ID NO: 19, a peptide linker that is 3 aa in length, and an IgG1 FcEM 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 that is 3 aa in length, and an IgG1 FcEM 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: 185 (P127). 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 (P127).

[0169] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 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 the amino acid sequence of SEQ ID NO: 5, a peptide linker that is 14 aa in length, and an IgG1 FcDL 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: 5, a peptide linker that is 14 aa in length, and an IgG1 FcDL 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 (P622). 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 (P622).

[0170] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 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 the amino acid sequence of SEQ ID NO: 5, a peptide linker that is 3 aa in length, and an IgG1 FcDL 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: 5, a peptide linker that is 3 aa in length, and an IgG1 FcDL 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: 187 (P624). 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 (P624).

[0171] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 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 the amino acid sequence of SEQ ID NO: 5, a peptide linker that is 3 aa in length, and an IgG1 FcY-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: 5, a peptide linker that is 3 aa in length, and an IgG1 FcY-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: 188 (P625). 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 (P625).

[0172] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 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:5, a peptide linker that is 3 aa in length, and an IgG1 FcYTE-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:5, a peptide linker that is 3 aa in length, and an IgG1 FcYTE-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 (P626). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 189 (P626).

[0173] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 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:5, a peptide linker that is 19 aa in length, and an IgG1 Fc-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:5, a peptide linker that is 19 aa in length, and an IgG1 Fc-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: 190 (P666). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 190 (P666).

[0174] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 39 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:5, a peptide linker that is 39 aa in length, and an IgG1 Fc-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:5, a peptide linker that is 39 aa in length, and an IgG1 Fc-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: 191 (P667). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 191 (P667).

[0175] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 14 aa in length, and an IgG2 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: 5, a peptide linker that is 14 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 5, a peptide linker that is 14 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P671). 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 (P671).

[0176] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 19 aa in length, and an IgG2 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: 5, a peptide linker that is 19 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 5, a peptide linker that is 19 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P672). 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 (P672).

[0177] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 39 aa in length, and an IgG2 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: 5, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 5, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P673). 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 (P673).

[0178] In some embodiments, the 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 F58E amino acid substitution, a peptide linker that is 3 aa in length, and an IgG2 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: 5, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 5, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P674). 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 (P674).

[0179] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 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:6, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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:6, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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: 196 (P683). 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 (P683).

[0180] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 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:6, a peptide linker that is 3 aa in length, and an IgG1 Fc-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:6, a peptide linker that is 3 aa in length, and an IgG1 Fc-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: 197 (P684). 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 (P684).

[0181] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 3 aa in length, and an IgG2 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:6, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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:6, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P685). 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 (P685).

[0182] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 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:6, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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:6, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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: 199 (P686). 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 (P686).

[0183] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 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:6, a peptide linker that is 14 aa in length, and an IgG1 Fc-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:6, a peptide linker that is 14 aa in length, and an IgG1 Fc-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: 200 (P687). 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 (P687).

[0184] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 39 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:6, a peptide linker that is 39 aa in length, and an IgG1 Fc-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:6, a peptide linker that is 39 aa in length, and an IgG1 Fc-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 (P688). 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 (P688).

[0185] In some embodiments, the 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 F58D amino acid substitution, a peptide linker that is 39 aa in length, and an IgG2 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:6, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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:6, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P689). 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 (P689).

[0186] In some embodiments, the 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 F58Y amino acid substitution, a peptide linker that is 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:7, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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:7, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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: 203 (P690). 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 (P690).

[0187] In some embodiments, the 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 F58Y amino acid substitution, a peptide linker that is 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:7, a peptide linker that is 3 aa in length, and an IgG1 Fc-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:7, a peptide linker that is 3 aa in length, and an IgG1 Fc-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 (P691). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 204 (P691).

[0188] In some embodiments, the 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 F58Y amino acid substitution, a peptide linker that is 3 aa in length, and an IgG2 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:7, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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:7, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P692). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 205 (P692).

[0189] In some embodiments, the 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 F58Y amino acid substitution, a peptide linker that is 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:7, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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:7, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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: 206 (P693). In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 206 (P693).

[0190] In some embodiments, the 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 F58Y amino acid substitution, a peptide linker that is 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:7, a peptide linker that is 14 aa in length, and an IgG1 Fc-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:7, a peptide linker that is 14 aa in length, and an IgG1 Fc-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 (P694). 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 (P694).

[0191] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Y amino acid substitution, a peptide linker that is 39 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:7, a peptide linker that is 39 aa in length, and an IgG1 Fc-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:7, a peptide linker that is 39 aa in length, and an IgG1 Fc-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: 208 (P695). 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 (P695).

[0192] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Y amino acid substitution, a peptide linker that is 39 aa in length, and an IgG2 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:7, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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:7, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P696). 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 (P696).

[0193] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 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: 8, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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: 8, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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 (P697). 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 (P697).

[0194] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 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: 8, a peptide linker that is 3 aa in length, and an IgG1 Fc-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: 8, a peptide linker that is 3 aa in length, and an IgG1 Fc-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 (P698). 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 (P698).

[0195] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 3 aa in length, and an IgG2 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: 8, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 8, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P699). 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 (P699).

[0196] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 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: 8, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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: 8, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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 (P700). 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 (P700).

[0197] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 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: 8, a peptide linker that is 14 aa in length, and an IgG1 Fc-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: 8, a peptide linker that is 14 aa in length, and an IgG1 Fc-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 (P701). 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 (P701).

[0198] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 39 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: 8, a peptide linker that is 39 aa in length, and an IgG1 Fc-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: 8, a peptide linker that is 39 aa in length, and an IgG1 Fc-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 (P702). 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 (P702).

[0199] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58K amino acid substitution, a peptide linker that is 39 aa in length, and an IgG2 Fc domain. In some embodiments, the 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: 8, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 8, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P703). 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 (P703).

[0200] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 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:9, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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:9, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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 (P704). 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 (P704).

[0201] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 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:9, a peptide linker that is 3 aa in length, and an IgG1 Fc-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:9, a peptide linker that is 3 aa in length, and an IgG1 Fc-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 (P705). 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 (P705).

[0202] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 3 aa in length, and an IgG2 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:9, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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:9, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P706). 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 (P706).

[0203] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 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:9, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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:9, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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 (P707). 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 (P707).

[0204] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 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:9, a peptide linker that is 14 aa in length, and an IgG1 Fc-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:9, a peptide linker that is 14 aa in length, and an IgG1 Fc-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 (P708). 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 (P708).

[0205] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 39 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:9, a peptide linker that is 39 aa in length, and an IgG1 Fc-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:9, a peptide linker that is 39 aa in length, and an IgG1 Fc-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 (P709). 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 (P709).

[0206] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58Q amino acid substitution, a peptide linker that is 39 aa in length, and an IgG2 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: 9, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 9, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P710). 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 (P710).

[0207] In some embodiments, the 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 F58W amino acid substitution, a peptide linker that is 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: 10, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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: 10, a peptide linker that is 3 aa in length, and an IgG1 Fc-EM 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 (P711). 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 (P711).

[0208] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58W amino acid substitution, a peptide linker that is 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: 10, a peptide linker that is 3 aa in length, and an IgG1 Fc-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: 10, a peptide linker that is 3 aa in length, and an IgG1 Fc-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 (P712). 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 (P712).

[0209] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58W amino acid substitution, a peptide linker that is 3 aa in length, and an IgG2 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: 10, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 10, a peptide linker that is 3 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P713). 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 (P713).

[0210] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58W amino acid substitution, a peptide linker that is 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: 10, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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: 10, a peptide linker that is 14 aa in length, and an IgG1 Fc-EM 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 (P714). 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 (P714).

[0211] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58W amino acid substitution, a peptide linker that is 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: 10, a peptide linker that is 14 aa in length, and an IgG1 Fc-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: 10, a peptide linker that is 14 aa in length, and an IgG1 Fc-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 (P715). 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 (P715).

[0212] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58W amino acid substitution, a peptide linker that is 39 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: 10, a peptide linker that is 39 aa in length, and an IgG1 Fc-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: 10, a peptide linker that is 39 aa in length, and an IgG1 Fc-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 (P716). 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 (P716).

[0213] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising an F58W amino acid substitution, a peptide linker that is 39 aa in length, and an IgG2 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: 10, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. In some embodiments, the 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: 10, a peptide linker that is 39 aa in length, and an IgG2 Fc domain monomer of SEQ ID NO: 157. 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 (P717). 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 (P717).

[0214] In some embodiments, the TGFβ superfamily ligand binding agent is a homodimeric polypeptide, wherein each polypeptide chain comprises, from N-terminus to C-terminus, an ActRIIB ECD comprising a G27D amino acid substitution, a peptide linker that is 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 that is 14 aa in length, and an IgG1 FcDL 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: 10, a peptide linker that is 14 aa in length, and an IgG1 FcDL 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 (P758). 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 (P758).

[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 an F58K amino acid substitution, a peptide linker that is 6 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: 8, a peptide linker that is 6 aa in length, and an IgG1 FcDL 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: 8, a peptide linker that is 6 aa in length, and an IgG1 FcDL 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 (P1153). 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 (P1153).

[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 an F58K amino acid substitution, a peptide linker that is 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:8, a peptide linker that is 10 aa in length, and an IgG1 FcDL 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:8, a peptide linker that is 10 aa in length, and an IgG1 FcDL 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 (P1154). 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 (P1154).

[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 an F58K amino acid substitution, a peptide linker that is 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:8, a peptide linker that is 19 aa in length, and an IgG1 FcDL 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:8, a peptide linker that is 19 aa in length, and an IgG1 FcDL 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 (P1155). 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 (P1155).

[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 an F58K amino acid substitution, a peptide linker that is 39 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:8, a peptide linker that is 39 aa in length, and an IgG1 FcDL 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:8, a peptide linker that is 39 aa in length, and an IgG1 FcDL 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 (P1156). 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 (P1156).

[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 an F58E amino acid substitution, a peptide linker that is 6 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:5, a peptide linker that is 6 aa in length, and an IgG1 FcDL 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:5, a peptide linker that is 6 aa in length, and an IgG1 FcDL 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 (P1163). 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 (P1163).

[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 an F58E amino acid substitution, a peptide linker that is 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:5, a peptide linker that is 10 aa in length, and an IgG1 FcDL 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:5, a peptide linker that is 10 aa in length, and an IgG1 FcDL 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 (P1164). 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 (P1164).

[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 and a C-terminal APT sequence comprising an F58E amino acid substitution, a peptide linker that is 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 and a C-terminal APT sequence comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, a peptide linker that is 14 aa in length, and an IgG1 FcDL 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 and a C-terminal APT sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 5, a peptide linker that is 14 aa in length, and an IgG1 FcDL 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 (P1168). 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 (P1168).

[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 an F58N amino acid substitution, a peptide linker that is 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 that is 14 aa in length, and an IgG1 FcDL 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 that is 14 aa in length, and an IgG1 FcDL 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 (P1213). 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 (P1213).

[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 an F58R amino acid substitution, a peptide linker that is 14 aa in length, and an IgG1 Fc domain monomer. In some embodiments,...

Claims

[Claim 1] A polypeptide comprising: a. an activin receptor type IIB (ActRIIB) ectodomain (ECD) variant; and b. a peptide linker comprising at least 10 amino acids; c. an Fc domain monomer; The polypeptide comprising: