METHODS OF USING ACTIVIN TYPE II RECEPTOR SIGNALING INHIBITORS

Abstract

The invention features methods of treating a transfusion dependent subject having myelofibrosis, a cytopenia associated with myelofibrosis, such as anemia, thrombocytopenia, or neutropenia, or receiving treatment with a cytopenia-associated myelofibrosis treatment by administering to the subject an activin receptor type II (ActRII) signaling inhibitor, optionally in combination with a cytopenia- associated myelofibrosis treatment. The ActRII signaling inhibitor may be an antibody that binds to an ActRII ligand, an ActRII antibody, or an ActRII ligand trap.

Description

[0001] METHODS OF USING ACTIVIN RECEPTOR TYPE II SIGNALING INHIBITORS

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 22, 2024, is named 51 184-054WO3_Sequence_Listing_1 1_22_24.xml and is 1 ,428,551 bytes in size.

[0004] BACKGROUND OF THE INVENTION

[0005] Myelofibrosis is a chronic myeloproliferative malignancy characterized by clonal proliferation of myeloid cells and megakaryocytic hyperplasia / dysplasia resulting in bone marrow fibrosis and osteosclerosis. It can present as a de novo disorder (primary myelofibrosis, PMF) or evolve from polycythemia vera (post-PV MF), essential thrombocythemia (post-ET MF), myelodysplastic syndrome (MDS), lupus, or other hematologic and solid tumors. Myeloproliferative neoplasms arise from a single somatically mutated hematopoietic stem cell progenitor that clonally expands and gives rise to virtually all myeloid cells and B and natural killer cells. It is characterized by bone marrow fibrosis, ineffective hematopoiesis, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, and shortened survival. Extensive scarring in the bone marrow in subjects with myelofibrosis can lead to severe anemia and a low number of platelets. Symptoms of myelofibrosis include fatigue, bone pain, easy bruising, easy bleeding, and fever. There are no curative medical therapies for patients with myelofibrosis, but JAK inhibitors, such as ruxolitinib (JAKAFIO / JAKAVI®), fedratinib (INREBIC®), and pacritinib (VONJO™) have been shown to reduce spleen volume and improve symptoms associated with myelofibrosis. However, JAK inhibitors interfere with normal hematopoiesis and treatment with ruxolitinib and fedratinib is complicated by the development of anemia and thrombocytopenia, which can lead to dose reductions and reduced adherence, thereby limiting the number of patients able to remain on JAK inhibitors. Patients with aggressive or high-risk myelofibrosis may require a blood transfusion or bone marrow transplant. Other treatment options include therapies that have known risks, such as androgen therapy and treatment with thalidomide or related medications. For patients with intermediate-risk myelofibrosis, treatment is typically directed at symptom management.

[0006] Accordingly, there exists a need for effective treatments for myelofibrosis-associated cytopenias and a new therapeutic approach to prevent or reduce the development of cytopenias in subjects treated with JAK inhibitors.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention features ActRII signaling inhibitors, including activin A antibodies, activin B antibodies, myostatin antibodies, GDF-11 antibodies, ActRII antibodies, and ActRII ligand traps (e.g., ActRIIA ligand traps, ActRIIB ligand traps, and ActRII chimera ligand traps, such as ActRIIA ligand traps including an extracellular ActRIIA variant). In some embodiments, an ActRII ligand trap includes an extracellular ActRII variant fused to the N- or C-terminus of an Fc domain, Fc domain monomer, or other moiety. Such moieties may be attached by amino acid or other covalent bonds and may increase stability of the polypeptide. An ActRII ligand trap including an extracellular ActRII variant fused to an Fc domain monomer may also form a dimer (e.g., a homodimer or heterodimer) through the interaction between two Fc domain monomers. The ActRII signaling inhibitors described herein may be used to treat a transfusion-dependent (TD) subject having or at risk of developing a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis, for example, by increasing hemoglobin levels, increasing hematocrit, increasing red blood cell count, promoting or increasing the maturation and / or differentiation of erythroid progenitors, increasing late-stage erythroid precursor maturation, recruiting early-stage progenitors into the erythroid lineage, increasing proerythroblast numbers, increasing reticulocytes, increasing early-stage erythroid precursor and / or progenitor numbers, promoting the progression of erythroid precursors and / or progenitors through erythropoiesis, increasing platelet levels (e.g., increasing platelet count), increasing neutrophil levels (e.g., increasing neutrophil count), reducing transfusion burden, and / or promoting transfusion independence. The ActRII signaling inhibitors of the invention may also be used to treat other features of myelofibrosis (e.g., splenomegaly or constitutional symptoms, such as symptoms assessed by MF-SAF-TSS score). The ActRII signaling inhibitor may be administered alone or in combination with a cytopenia-associated myelofibrosis treatment, such as ruxolitinib (JAKAFIO / JAKAVI®), fedratinib (INREBIC®), pacritinib (VONJO™), and imetelstat. The ActRII signaling inhibitor can also be administered in combination with a cytopenia- associated myelofibrosis treatment to a TD subject to treat polycythemia vera, steroid-refractory graft versus host disease, or chronic graft-versus-host disease or to treat a cytopenia in a subject having these conditions. Combination therapy can also be used to mitigate the adverse reactions associated with treatment with a cytopenia-associated myelofibrosis treatment and can improve treatment adherence, treatment duration, maintain dose intensity, or increase the dose of a cytopenia-associated myelofibrosis treatment, or decrease episodes of cytopenia, transfusion burden, bleeding events, infections, and treatment interruptions or discontinuations for a cytopenia-associated myelofibrosis treatment.

[0009] Exemplary embodiments of the invention are described in the enumerated paragraphs below.

[0010] E1 . A method of treating a transfusion-dependent subject having or at risk of developing a cytopenia associated with myelofibrosis, the method comprising the step of administering to the subject a therapeutically effective amount of an ActRII signaling inhibitor.

[0011] E2. A method of treating a transfusion-dependent subject having myelofibrosis, the method comprising the step of administering to the subject a therapeutically effective amount of an ActRII signaling inhibitor.

[0012] E3. The method of E2, wherein the subject has or is at risk of developing a cytopenia.

[0013] E4. The method of any one of E1-E3, wherein the subject discontinued treatment with a JAK inhibitor

[0014] (i.e., prior to administration of the ActRII signaling inhibitor).

[0015] E5. The method of E4, wherein the subject had relapsed disease following treatment with the JAK inhibitor.

[0016] E6. The method of E4, wherein the subject is refractory to treatment with the JAK inhibitor.

[0017] E7. The method of E4, wherein the subject is intolerant to treatment with a JAK inhibitor or no longer meets the risk / benefit ratio to continue treatment with the JAK inhibitor.

[0018] E8. The method of any one of E1-E3, wherein the subject is ineligible for treatment with a JAK inhibitor. E9. The method of any one of E1-E3, wherein the administering of the ActRII signaling inhibitor is in combination with administering a cytopenia-associated myelofibrosis treatment (e.g., an effective amount of a cytopenia-associated myelofibrosis treatment).

[0019] E10. The method of E9, wherein the administering in combination is performed after the subject has been identified (e.g., diagnosed) as having a cytopenia (e.g., a cytopenia developed during treatment with the cytopenia-associated myelofibrosis treatment, e.g., the ActRII signaling inhibitor is administered to a subject already undergoing treatment with the cytopenia-associated myelofibrosis treatment).

[0020] E11 . The method of E9, wherein the administering in combination is performed before the subject develops a cytopenia (e.g., to prevent the development of a cytopenia or reduce the risk of developing a cytopenia).

[0021] E12. A method of treating a transfusion-dependent subject receiving treatment with a cytopenia- associated myelofibrosis treatment, the method comprising the step of administering in combination to the subject an effective amount of a cytopenia-associated myelofibrosis treatment and an ActRII signaling inhibitor.

[0022] E13. The method of E12, wherein the subject has or is at risk of developing a cytopenia.

[0023] E14. The method of E12 or E13, wherein the subject has myelofibrosis, polycythemia vera (e.g., an adult subject who has had an inadequate response to or is intolerant of hydroxyurea), steroid- refractory graft versus host disease (e.g., acute graft-versus-host disease), or chronic graft- versus-host disease (e.g., chronic graft versus host disease after failure of one or two lines of systemic therapy in an adult subject or a pediatric subject 12 years of age or older).

[0024] E15. The method of any one of E9-E14, wherein the method improves adherence to treatment with the cytopenia-associated myelofibrosis treatment.

[0025] E16. The method of any one of E9-E14, wherein the method allows treatment with a cytopenia- associated myelofibrosis treatment to resume (e.g., after a discontinuation).

[0026] E17. The method of any one of E9-E16, wherein the method allows the dose of the cytopenia- associated myelofibrosis treatment to be increased (e.g., the subject can take a higher dose when the two agents are co-administered than when the cytopenia-associated myelofibrosis treatment is administered alone).

[0027] E18. The method of any one of E9-E16, wherein the method allows the dose of the cytopenia- associated myelofibrosis treatment to be maintained (e.g., the subject does not need to reduce the dose of the cytopenia-associated myelofibrosis treatment when the two agents are coadministered or requires fewer or smaller dose reductions than when the cytopenia-associated myelofibrosis treatment is administered alone).

[0028] E19. The method of any one of E9-E18, wherein the method allows treatment duration with the cytopenia-associated myelofibrosis treatment to be increased (e.g., the subject can continue to take the cytopenia-associated myelofibrosis treatment for a longer period of time when the two agents are co-administered than when the cytopenia-associated myelofibrosis treatment is administered alone).

[0029] E20. The method of any one of E9-E19, wherein the method decreases episodes of cytopenia associated with the cytopenia-associated myelofibrosis treatment. E21 . The method of any one of E9-E20, wherein the method decreases bleeding events.

[0030] E22. The method of any one of E9-E21 , wherein the method decreases infections.

[0031] E23. The method of any one of E9-E22, wherein the method decreases treatment interruptions or discontinuations for the cytopenia-associated myelofibrosis treatment.

[0032] E24. The method of any one of E9-E23, wherein the cytopenia-associated myelofibrosis treatment is a JAK inhibitor or Imetelstat.

[0033] E25. The method of any one of E4-E8 and E24, wherein the JAK inhibitor is ruxolitinib, fedratinib, or pacritinib.

[0034] E26. The method of E25, wherein the JAK inhibitor is ruxolitinib.

[0035] E27. The method of any one of E1-E11 and E14-E26, wherein the myelofibrosis is primary myelofibrosis (PMF).

[0036] E28. The method of any one of E1-E11 and E14-E26, wherein the myelofibrosis is post-essential thrombocythemia myelofibrosis (post-ET MF).

[0037] E29. The method of any one of E1-E11 and E14-E26, wherein the myelofibrosis is post-polycythemia vera myelofibrosis (post-PV MF).

[0038] E30. The method of any one of E1-E11 and E14-E29, wherein the myelofibrosis is intermediate- or high-risk myelofibrosis (e.g., according to the dynamic international prognostic scoring system (DIPSS)).

[0039] E31 . The method of E30, wherein the myelofibrosis is intermediate-1 risk myelofibrosis.

[0040] E32. The method of E30, wherein the myelofibrosis is intermediate-2 risk myelofibrosis.

[0041] E33. The method of E30, wherein the myelofibrosis is high-risk myelofibrosis.

[0042] E34. The method of any one of E1-E33, wherein the method reduces osteosclerosis.

[0043] E35. The method of any one of E1-E34, wherein the method leads to a reduction in transfusion burden during a treatment period (e.g., a reduction in RBC units transfused during a treatment period of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 20 weeks, 24, weeks, 26 weeks, 1 year, 2 years or more with an ActRII signaling inhibitor compared to baseline RBC transfusion requirements, e.g., compared to baseline RBC transfusion requirements in the 12 weeks immediately preceding the first dose of the ActRII signaling inhibitor).

[0044] E36. The method of any one of E1-E35, wherein the method promotes transfusion independence (e.g., transfusion independence for at least 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 20 weeks, 24 weeks, 26 weeks, 1 year, 2 years or more during treatment with an ActRII signaling inhibitor compared to pretreatment transfusion data).

[0045] E37. The method of any one of E1-E36, wherein the method reduces splenomegaly (e.g., reduces spleen volume).

[0046] E38. The method of any one of E1-E37, wherein the method reduces bone marrow fibrosis.

[0047] E39. The method of any one of E1-E38, wherein the method improves constitutional symptoms (e.g., as assessed using MF-SAF-TSS, such as a reduction in MF-SAF-TSS).

[0048] E40. The method of any one of E1 , E3-E11 , and E13-E39, wherein the cytopenia is anemia.

[0049] E41 . The method of any one of E1 , E3-E11 , and E13-E40, wherein the cytopenia is thrombocytopenia.

[0050] E42. The method of any one of E1 , E3-E11 , and E13-E41 , wherein the cytopenia is neutropenia. E43. The method of any one of E1-E42, wherein the ActRII signaling inhibitor is an activin A antibody or an antigen binding fragment thereof.

[0051] E44. The method of E43, wherein the activin A antibody is garetosmab.

[0052] E45. The method of E43, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 and a light chain variable region (LCVR) sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 (e.g., an HCVR sequence in Table 1 and an LCVR sequence in Table 1 , such as an HCVR sequence and an LCVR sequence from the same row of Table 1).

[0053] E46. The method of E43 or E45, wherein the activin A antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 2 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 2).

[0054] E47. The method of any one of E1-E42, wherein the ActRII signaling inhibitor is a myostatin antibody or an antigen binding fragment thereof.

[0055] E48. The method of E47, wherein the myostatin antibody is domagrozumab, landogrozumab, trevogrumab, or SRK-015.

[0056] E49. The method of E47, wherein the myostatin antibody or an antigen binding fragment thereof has a HCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4 and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4 (e.g., a HCVR sequence in Table 4 and a LCVR sequence in Table 4, such as an HCVR sequence and an LCVR sequence from the same row of Table 4 or an HCVR sequence of any one of SEQ ID NOs: 448-476 and an LCVR sequence of any one of SEQ ID NOs: 477-486).

[0057] E50. The method of E47 or E49, wherein the myostatin antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 5, Table 6, or Table 7 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 5).

[0058] E51 . The method of any one of E47, E49, and E50, wherein the myostatin antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 8 (e.g., a heavy chain and light chain sequence from the same row of Table 8).

[0059] E52. The method of any one of E1-E42, wherein the ActRII signaling inhibitor is an ActRII antibody or an antigen binding fragment thereof.

[0060] E53. The method of E52, wherein the ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861 .

[0061] E54. The method of E52, wherein the ActRII antibody or an antigen binding fragment thereof has a

[0062] HCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 9 and a LCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 9 (e.g., a HCVR sequence in Table 9 and a LCVR sequence in Table 9, such as an HCVR sequence and an LCVR sequence from the same row of Table 9).

[0063] E55. The method of E52 or E54, wherein the ActRII antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 10 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 10).

[0064] E56. The method of any one of E52, E54, and E55, wherein the ActRII antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 1 1 (e.g., a heavy chain and light chain sequence from the same row of Table 11).

[0065] E57. The method of any one of E1-E42, wherein the ActRII signaling inhibitor is an ActRII ligand trap. E58. The method of E57, wherein the ActRII ligand trap is an ActRIIA ligand trap.

[0066] E59. The method of E58, wherein the ActRIIA ligand trap is a composition of Table 20 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 20).

[0067] E60. The method of E59, wherein the composition of Table 20 is a polypeptide comprising an extracellular activin receptor type IIA (ActRIIA) variant, the variant having a sequence of GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X10X11X12X13X14HCX15ATWX16NISG SIEIVX17X18GCX19X20X21DX22NCYDRTDCVEX23X24X25X26PX27VYFCCCEGNMCNEKFSYFPEME VTQPTS (SEQ ID NO: 1), wherein Xi is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; Xe is R or A; X7 is P or R; Xs is Y or E; X9 is D or E; X10 is K or Q; Xu is D or A; X12 is K or A; X13 is R or A; X14 is R or L; X15 is F or Y; X16 is K, R, or A; X17 is K, A, Y, F, or I; Xis is Q or K; X19 is W or A; X20 is L or A; X21 is D, K, R, A, F, G, M, N, or I; X22 is I, F, or A; X23 is K or T; X24 is K or E; X25 is D or E; X26 is S or N; and X27 is E or Q, and wherein the variant does not have the sequence of SEQ ID NO: 73.

[0068] E61 . The method of E60, wherein Xi is F or Y; X2 is Y; X4 is L; X5 is D or E; X7 is P or R; Xs is E; X9 is

[0069] E; X10 is K or Q; X14 is L; X15 is F or Y; X16 is K or R; Xis is K; X22 is I or F; X23 is T; X24 is K or E; X25 is E; X26 is N; and X27 is Q.

[0070] E62. The method of E61 , wherein Xi is F or Y; X2 is Y; X3 is E; X4 is L; X5 is D or E; Xe is R; X7 is P or

[0071] R; Xs is E; X X17 is K; Xis and X27 is Q.

[0072] E63. The method of E61 or E62, wherein Xi is F and X10 is K.

[0073] E64. The method of E60, wherein the wherein the variant has the sequence of any one of SEQ ID

[0074] NOs: 6-72.

[0075] E65. The method of E64, wherein the variant has the sequence of SEQ ID NO: 69. E66. The method of any one of E60-E65, wherein the polypeptide further includes an Fc domain monomer fused to the C-terminus of the polypeptide (e.g., the C-terminus of the ActRIIA variant) by way of a linker.

[0076] E67. The method of E66, wherein the polypeptide has the sequence of SEQ ID NO: 80.

[0077] E68. The method of E66 or E67, wherein the polypeptide is in the form of a dimer (e.g., a homodimer).

[0078] E69. The method of E58, wherein the ActRIIA ligand trap comprises an extracellular portion of wildtype ActRIIA (e.g., SEQ ID NO: 73 or SEQ ID NO: 729).

[0079] E70. The method of E58, wherein the ActRIIA ligand trap is sotatercept.

[0080] E71 . The method of E57, wherein the ActRII ligand trap is an ActRIIB ligand trap.

[0081] E72. The method of E71 , wherein the ActRIIB ligand trap comprises an extracellular portion of wildtype ActRIIB (e.g., SEQ ID NO: 74 or a portion thereof).

[0082] E73. The method of E71 , wherein the ActRIIB ligand trap is BIIB110, ALG-802, luspatercept, ramatercept, or ACE-2494.

[0083] E74. The method of E71 , wherein the ActRIIB ligand trap is a composition of Table 21 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 21).

[0084] E75. The method of E71 , wherein the ActRIIB ligand trap comprises the sequence of any one of SEQ ID NOs: 745-750 (e.g., the sequence of any one of SEQ ID NOs: 745-750 fused to a moiety, such as an Fc domain or an Fc domain monomer, by way of a linker).

[0085] E76. The method of E57, wherein the ActRII ligand trap is an ActRII chimera ligand trap.

[0086] E77. The method of E76, wherein the ActRII chimera ligand trap is a composition of Table 22 or Table

[0087] 23 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 22 or Table 23).

[0088] E78. The method of any one of E1-E42, wherein the ActRII signaling inhibitor is an activin B antibody or an antigen binding fragment thereof.

[0089] E79. The method of E78, wherein the activin B antibody or an antigen binding fragment thereof has a HCVR having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 494 and a LCVR having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 495.

[0090] E80. The method of any one of E1-E42, wherein the ActRII signaling inhibitor is a GDF-11 antibody or an antigen binding fragment thereof.

[0091] E81 . The method of any one of E1 , E3-E11 , and E13-E80, wherein the subject is identified (e.g., diagnosed) as having a cytopenia prior to administration of the ActRII signaling inhibitor.

[0092] E82. The method of any one of E1 , E3-E11 , and E13-E80, wherein the method further comprises identifying the subject as having a cytopenia prior to administration of the ActRII signaling inhibitor.

[0093] E83. The method of any one of E1-E82, wherein the method further comprises evaluating osteosclerosis, spleen volume, bone marrow fibrosis, platelet parameters (e.g., platelet number or platelet volume), red cell parameters e.g., RBC count, reticulocyte count, hematocrit levels, or hemoglobin levels), neutrophil levels, transfusion burden, and / or constitutional symptoms (e.g., using MF-SAF-TSS) before administration of the ActRII signaling inhibitor. E84. The method of any one of E1-E83, wherein the method further comprises evaluating osteosclerosis, spleen volume, bone marrow fibrosis, platelet parameters (e.g., platelet number or platelet volume), red cell parameters (e.g., RBC count, reticulocyte count, hematocrit levels, or hemoglobin levels), neutrophil levels, transfusion burden, and / or constitutional symptoms after administration of the ActRII signaling inhibitor.

[0094] E85. The method of any one of E1-E84, wherein the method leads to an increase in hemoglobin of >1 .5 g / dL (e.g., an increase in hemoglobin of >1 .5 g / dL for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 20 weeks, 24, weeks, 26 weeks, 1 year, 2 years or more during treatment with an ActRII signaling inhibitor compared to baseline pretreatment measurements).

[0095] E86. The method of any one of E1-E85, wherein the ActRII signaling inhibitor is administered in an amount sufficient to increase red blood cell levels, increase hemoglobin levels, increase red blood cell production, increase red blood cell count, increase hematocrit, reduce transfusion burden, promote transfusion independence, increase mean corpuscular volume, increase mean corpuscular hemoglobin, increase reticulocyte cell hemoglobin, increase erythropoietin levels, increase thrombopoietin levels, increase the maturation and / or differentiation of erythroid progenitors (e.g., early- and / or late-stage erythroid progenitors), increase late-stage erythroid precursor maturation, recruit early-stage progenitors into the erythroid lineage, increase reticulocytes, increase proerythroblast numbers, reduce the accumulation of red blood cell progenitor cells, increase the number of early-stage erythroid precursors and / or progenitors, promote the progression of erythroid precursors and / or progenitors through erythropoiesis, treat anemia, increase platelet levels, increase platelet volume, increase immature platelet fraction, increase proplatelets, increase platelet production, increase platelet count, increase or induce megakaryocyte differentiation and / or maturation, increase megakaryocyte progenitor renewal, reduce the accumulation of platelet progenitor cells, improve blood clotting, reduce bleeding events, reduce bleeding in the skin, treat thrombocytopenia, increase neutrophil levels, increase neutrophil production, increase neutrophil count, increase or induce the differentiation and / or maturation of progenitor cells into neutrophils, treat neutropenia, reduce susceptibility to infection, affect myostatin, activin A, activin B, and / or BMP9 signaling in the subject, or reduce or inhibit the binding of activin A, activin B, and / or myostatin to their receptors (e.g., their endogenous receptors).

[0096] E87. The method of any one of E1-86, wherein the ActRII signaling inhibitor is administered in an amount sufficient to reduce spleen volume, reduce bone marrow fibrosis, reduce osteosclerosis, improve bone marrow fibrosis grade, or improve constitutional symptoms (e.g., reduce MF-SAF- TSS).

[0097] E88. The method of any one of E1-E87, wherein the method does not cause a vascular complication in the subject.

[0098] E89. The method of E88, wherein the method does not increase vascular permeability or leakage. E90. The method of any one of E1-E89, wherein the subject is a human. Definitions

[0099] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the invention. Terms such as "a", "an," and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0100] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0101] As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.

[0102] As used herein, “administration” refers to providing or giving a subject a therapeutic agent (e.g., an ActRII signaling inhibitor described herein), by any effective route. Exemplary routes of administration are described herein below.

[0103] The term “antibody” is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0104] “Antibody fragments” include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0105] As used herein, the term “extracellular activin receptor type 11 A (ActRIIA) variant” refers to a peptide including a soluble, extracellular portion of the single transmembrane receptor, ActRIIA, that has at least one amino acid substitution relative to a wild-type extracellular ActRIIA (e.g., bold portion of the sequence of SEQ ID NO: 75 shown below). The sequence of the wild-type, human ActRIIA precursor protein is shown below (SEQ ID NO: 75), in which the signal peptide is italicized and the extracellular portion is bold.

[0106] Wild-type, human ActRIIA precursor protein (SEQ ID NO: 75):

[0107] / WGAAAKLAFAI / H. / SCSSGAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRR HCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSY FPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQ DPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEV YSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAET MARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGD THGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYML PFEEEIGQHPSLEDMQEVWHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARL SAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL

[0108] An extracellular ActRIIA variant may have a sequence of any one of SEQ ID NOs: 1-72. In particular embodiments, an extracellular ActRIIA variant has a sequence of any one of SEQ ID NOs: 6-72 (Table 13). In some embodiments, an extracellular ActRIIA variant may have at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 73).

[0109] As used herein, the terms “extracellular activin receptor type 11 B (ActRIIB) variant” and “ActRIIB variant” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor, ActRIIB, that has at least one amino acid substitution relative to a wild-type extracellular ActRIIB (e.g., bold portion of the sequence of SEQ ID NO: 829 shown below). The sequence of the wild-type, human ActRIIB is shown below (SEQ ID NO: 829), in which the signal peptide is italicized and the extracellular portion is bold.

[0110] Wild-type human ActRIIB (SEQ ID NO: 829):

[0111] / WTAPLWALALLH / GSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCY ASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAG GPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPP PSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMK HENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSY LHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVG TRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQ HPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSL IRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

[0112] An extracellular ActRIIB variant may have a sequence of any one of SEQ ID NOs: 730-750. In particular embodiments, an extracellular ActRIIB variant has a sequence of any one of SEQ ID NOs: 731 - 744 (Table 15). In some embodiments, an extracellular ActRIIB variant may have at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIB (SEQ ID NO: 74). The extracellular ActRIIB variant may also have an N-terminal truncation of 1-7 amino acids relative to the extracellular portion of ActRIIB.

[0113] As used herein, the terms “extracellular activin receptor type II (ActRII) chimera,” “extracellular ActRII chimera,” and “ActRII chimera” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor ActRIIB and a soluble, extracellular portion of the single transmembrane receptor ActRIIA. In some embodiments, the ActRII chimeras described herein result from joining an N- terminal portion of extracellular ActRIIB to a C-terminal portion of extracellular ActRIIA such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA). The extracellular ActRII chimera may also include one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIB compared to a wild-type extracellular ActRIIB (e.g., bold portion of the sequence of SEQ ID NO: 829 shown above), and one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIA compared to a wild-type extracellular ActRIIA (e.g., bold portion of the sequence of SEQ ID NO: 75 shown above). In other embodiments, the ActRII chimeras result from the substitution of one or more amino acid sequence corresponding a p-sheet from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA) and / or from the substitution of one or more intervening sequence (e.g., a sequence between the p-sheets) from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA). For example, an ActRII chimera may be produced by replacing one or more amino acid sequence corresponding to a p-sheet in ActRIIB with an amino acid sequence corresponding to the p-sheet from ActRIIA. The extracellular ActRII chimera may also have an N-terminal truncation of 1-9 amino acids relative to the extracellular portion of ActRIIB or ActRIIA. The sequences of wild-type, human ActRIIB (SEQ ID NO: 829) and wild-type, human ActRIIA (SEQ ID NO: 75) are shown in the definitions above, in which the signal peptide is italicized and the extracellular portion is bold. An extracellular ActRII chimera may have the sequence of any one of SEQ ID NOs: 751-793 or 1029-1059. In particular embodiments, an extracellular ActRII chimera has the sequence of any one of SEQ ID NOs: 772-793 (Table 17).

[0114] As used herein, the term “extracellular activin receptor type II (ActRII) variant” refers to an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera described herein.

[0115] As used herein, the term “N-terminal truncation” refers to a deletion of 1-7 amino acids (e.g., 1 , 2, 3, 4, 5, 6, or 7 amino acids) from the N-terminus of an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731-750)) or a deletion of 1-9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) from the N-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751-793 or 1029-1059 (e.g., SEQ ID NOs: 772-793)). The N-terminal truncation can remove amino acids up to two amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimeras and the two amino acids before the first cysteine (RE) are retained in the N-terminally truncated ActRIIB variants).

[0116] As used herein, the term “cytopenia-associated myelofibrosis treatment” refers to a drug that is either approved for the treatment of myelofibrosis or that is in clinical development for the treatment of myelofibrosis and that has as an adverse reaction the development of a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia).

[0117] As used herein, the term “linker” refers to a linkage between two elements, e.g., peptides or protein domains. An ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety. The moiety may increase stability or improve pharmacokinetic properties of the polypeptide. The moiety (e.g., Fc domain monomer, Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin) may be fused to the polypeptide by way of a linker. A linker can be a covalent bond or a spacer. The term “bond” refers to a chemical bond, e.g., an amide bond or a disulfide bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. The term “spacer” refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence) occurring between two elements, e.g., peptides or protein domains, to provide space and / or flexibility between the two elements. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to the spaced peptides via the polypeptide backbone). The formation of disulfide bonds, e.g., between two hinge regions that form an Fc domain, is not considered a linker. As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers. An Fc domain has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) to a human Fc domain that includes at least a CH2 domain and a CH3 domain. An Fc domain monomer includes second and third antibody constant domains (CH2 and CH3). In some embodiments, the Fc domain monomer also includes a hinge domain. An Fc domain does not include any portion of an immunoglobulin that is capable of acting as an antigen-recognition region, e.g., a variable domain or a complementarity determining region (CDR). In the wild-type Fc domain, the two Fc domain monomers dimerize by the interaction between the two CH3 antibody constant domains, as well as one or more disulfide bonds that form between the hinge domains of the two dimerizing Fc domain monomers. In some embodiments, an Fc domain may be mutated to lack effector functions, typical of a “dead Fc domain.” In certain embodiments, each of the Fc domain monomers in an Fc domain includes amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and an Fey receptor. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. An Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Additionally, an Fc domain can be an IgG subtype (e.g., lgG1 , lgG2a, lgG2b, lgG3, or lgG4). The Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.

[0118] As used herein, the term “albumin-binding peptide” refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and functions to bind serum albumin. An albumin-binding peptide can be of different origins, e.g., human, mouse, or rat. In some embodiments, an albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 83).

[0119] 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, e.g., membrane-spanning receptor proteins such as integrins and extracellular matrix components such as collagens and fibrins. In some embodiments, a fibronectin domain is a fibronectin type III domain (SEQ ID NO: 82) having amino acids 610-702 of the sequence of UniProt ID NO: P02751 . In other embodiments, a fibronectin domain is an adnectin protein.

[0120] As used herein, the term “human serum albumin” refers to the albumin protein present in human blood plasma. Human serum albumin is the most abundant protein in the blood. It constitutes about half of the blood serum protein. In some embodiments, a human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 81).

[0121] As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell, e.g., a human red blood cell, platelet, neutrophil, or muscle cell).

[0122] As used herein, the term “fused” is used to describe the combination or attachment of two or more elements, components, or protein domains, e.g., peptides or polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., amide bonds. For example, two single peptides in tandem series can be fused to form one contiguous protein structure, e.g., a polypeptide, through chemical conjugation, a chemical bond, a peptide linker, or any other means of covalent linkage. In some embodiments of an ActRII ligand trap described herein, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused in tandem series to the N- or C-terminus of a moiety (e.g., Fc domain monomer (e.g., the sequence of SEQ ID NO: 97, SEQ ID NO: 84, or SEQ ID NO: 79), an Fc domain, an albumin-binding peptide (e.g., the sequence of SEQ ID NO: 83), a fibronectin domain (e.g., the sequence of SEQ ID NO: 82), or a human serum albumin (e.g., the sequence of SEQ ID NO: 81)) by way of a linker. For example, an extracellular ActRIIA variant is fused to a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin) by way of a peptide linker, in which the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIA variant through a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the moiety (e.g., Fc domain monomer, Fc domain, albumin-binding peptide, fibronectin domain, or human serum albumin) through a chemical bond, e.g., a peptide bond.

[0123] As used herein, the term “C-terminal extension” refers to the addition of one or more amino acids to the C-terminus of a polypeptide including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) or at the C- terminus of a an extracellular ActRI I chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751-793 or 1029-1059 (e.g., SEQ ID NOs: 772-793)). The C-terminal extension can be one or more amino acids, such as 1-6 amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids). The C-terminal extension may include amino acids from the corresponding position of wild-type ActRIIA (for an ActRIIA variant) or from the corresponding position of wild-type ActRIIA or ActRIIB (for an ActRII chimera). Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C- terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 78) (a six amino acid C-terminal extension). Any amino acid sequence that does not disrupt the activity of the polypeptide can be used. SEQ ID NO: 71 , which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NP, and SEQ ID NO: 72, which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NPVTPK (SEQ ID NO: 78), represent two of the possible ways that a polypeptide described herein can be modified to include a C-terminal extension.

[0124] As used herein, the term “percent (%) identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence, e.g., an extracellular ActRIIA variant, that are identical to the amino acid (or nucleic acid) residues of a reference sequence, e.g., a wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0125] 100 x (fraction of A / B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0126] In particular embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0127] As used herein, the term “serum half-life” refers to, in the context of administering a therapeutic protein to a subject, the time required for plasma concentration of the protein in the subject to be reduced by half. The protein can be redistributed or cleared from the bloodstream, or degraded, e.g., by proteolysis. Serum half-life comparisons can be made by comparing the serum half-life of Fc fusion proteins.

[0128] As used herein, the term “affinity” or “binding affinity” refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum total of non-covalent interactions between a molecule and its binding partner, such as an extracellular ActRIIA variant and BMP9 or activin A. Unless indicated otherwise, binding affinity refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair. The binding affinity between two molecules is commonly described by the dissociation constant (KD) or the affinity constant (KA). TWO molecules that have low binding affinity for each other generally bind slowly, tend to dissociate easily, and exhibit a large KD. TWO molecules that have high affinity for each other generally bind readily, tend to remain bound longer, and exhibit a small KD. The KD of two interacting molecules may be determined using methods and techniques well known in the art, e.g., surface plasmon resonance. KD is calculated as the ratio of kotr / kon.

[0129] As used herein, the phrase “affecting myostatin, activin A, activin B, and / or BMP9 signaling” means changing the binding of myostatin, activin A, activin B, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII (e.g., ActRIIA, e.g., endogenous ActRIIA). In some embodiments, a polypeptide including an extracellular ActRIIA variant described herein reduces or inhibits the binding of myostatin, activin A, activin B, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII (e.g., ActRIIA, e.g., endogenous ActRIIA). As used herein, the terms “increasing” and “decreasing” refer to modulating resulting in, respectively, greater or lesser amounts, of function, expression, or activity of a metric relative to a reference. For example, subsequent to administration of a polypeptide of the invention including an extracellular ActRIIA variant in a method described herein, the amount of a marker of a metric (e.g., hemoglobin levels, red blood cell count, hematocrit, reticulocyte count, platelet count, or transfusion burden) as described herein may be increased or decreased in a subject relative to the amount of the marker prior to administration. Generally, the metric is measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least one week, one month, 3 months, or 6 months, after a treatment regimen has begun.

[0130] As used herein, the terms “increase red blood cell levels” and “promote red blood cell formation” refer to clinically observable metrics, such as hematocrit, red blood cell counts, and hemoglobin measurements, and are intended to be neutral as to the mechanism by which such changes occur. The terms “red blood cell formation” and “red blood cell production” refer to the generation of red blood cells, such as the process of erythropoiesis in which red blood cells are produced in the bone marrow.

[0131] As used herein, the term "anemia" refers to any abnormality in hemoglobin or red blood cells that leads to reduced oxygen levels in the blood. Anemia can be associated with abnormal production, processing, or performance of erythrocytes and / or hemoglobin. The term anemia refers to any reduction in the number of red blood cells and / or level of hemoglobin in blood relative to normal blood levels. For example, a subject having a hemoglobin level <10 g / dL or receiving red blood cell (RBC) transfusions can be identified as having anemia.

[0132] As used herein, the terms “increase platelet levels” and “promote platelet formation” refer to clinically observable metrics, such as platelet counts, and are intended to be neutral as to the mechanism by which such changes occur. The terms “platelet formation” and “platelet production” refer to the generation of platelets, such as the process in which platelets are produced from megakaryocytes.

[0133] As used herein, the terms “increase neutrophil levels” and “promote neutrophil formation” refer to clinically observable metrics, such as neutrophil counts, and are intended to be neutral as to the mechanism by which such changes occur. The terms “neutrophil formation” and “neutrophil production” refer to the generation of neutrophils such as the process in which neutrophils are produced in the bone marrow.

[0134] As used herein, the term "thrombocytopenia" refers to a condition in which the blood contains a lower than normal number of platelets, which may be due to a deficiency in platelet production, accumulation of platelets within an enlarged spleen, or the destruction of platelets. Normal blood platelet levels range from about 150,000 to 450,000 per microliter blood in humans. A platelet count of less than 150,000 platelets per microliter is lower than normal. Bleeding can occur after a relatively minor injury if the platelet count falls below 50,000 platelets per microliter of blood, and serious bleeding may occur without any recognized injury if the platelet count falls below 10,000 to 20,000 platelets per microliter of blood.

[0135] As used herein, the term "neutropenia" refers to a condition in which the blood contains an abnormally low number of neutrophils. The typical lower limit of the neutrophil count is about 1500 cells per microliter of blood. Below this level, the risk of infection increases. Neutropenia severity is classified as: mild (1000 to 1500 neutrophils per microliter of blood), moderate (500 to 1000 neutrophils per microliter of blood), and severe (below 500 neutrophils per microliter of blood). Neutropenia has many causes, but they typically fall into two main categories: destruction or depletion of neutrophils faster than the bone marrow can produce new neutrophils, or reduced production of neutrophils in the bone marrow.

[0136] As used herein, the term “transfusion-dependent” (TD) is defined as receiving 6 or more (e.g., 6, 7, 8, 9, 10, or more) RBC units in the 12 weeks prior to first dose with an ActRII signaling inhibitor described herein, with at least one transfusion event in the 4 weeks preceding first dose. This is based on IWG 2013 criteria (Teferri et al., Blood, 2013).

[0137] As used herein, the term “non-transfusion-dependent” (non-TD) is defined as receiving less than 6 (e.g., 5, 4, 3, or fewer) RBC units in the12 weeks prior to first dose with an ActRII signaling inhibitor described herein. The non-TD subjects evaluated in the studies described herein also had baseline hemoglobin < 10 g / dL.

[0138] As used herein, the term “ineffective hematopoiesis” refers to the failure to produce fully mature hematopoietic cells (e.g., the failure to produce red blood cells, platelets, and neutrophils). Ineffective hematopoiesis may be due to single or multiple defects, such as abnormal proliferation and / or differentiation of progenitor cells (e.g., an excessive production of progenitors that are unable to complete differentiation), that can lead to a hyperproliferation or a shortage of progenitor cells.

[0139] As used herein, the terms “erythropoiesis stimulating agent” and “ESA” refer to a class of drugs that act on the proliferation stage of red blood cell development by expanding the pool of early-stage progenitor cells. Examples of erythropoiesis-stimulating agents are epoetin alfa and darbepoetin alfa.

[0140] As used herein, the term “vascular complication” refers to a vascular disorder or any damage to the blood vessels, such as damage to the blood vessel walls. Damage to the blood vessel walls may cause an increase in vascular permeability or leakage. The term “vascular permeability or leakage” refers to the capacity of the blood vessel walls to allow the flow of small molecules, proteins, and cells in and out of blood vessels. An increase in vascular permeability or leakage may be caused by an increase in the gaps (e.g., an increase in the size and / or number of the gaps) between endothelial cells that line the blood vessel walls and / or thinning of the blood vessel walls.

[0141] As used herein, the term “polypeptide” describes a single polymer in which the monomers are amino acid residues which are covalently conjugated together through amide bonds. A polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced.

[0142] As used herein, the term “homodimer” refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. The two identical monomers may form a homodimer by covalent bonds or non-covalent bonds. For example, an Fc domain may be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein including an extracellular ActRIIA variant fused to an Fc domain monomer may form a homodimer through the interaction of two Fc domain monomers, which form an Fc domain in the homodimer.

[0143] As used herein, the term “heterodimer” refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. The two monomers may form a heterodimer by covalent bonds or non-covalent bonds. For example, a polypeptide described herein including an extracellular ActRIIA variant fused to an Fc domain monomer may form a heterodimer through the interaction of two Fc domain monomers, each fused to a different ActRIIA variant, which form an Fc domain in the heterodimer.

[0144] As used herein, the term “host cell” refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express proteins from their corresponding nucleic acids. The nucleic acids are typically included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). A host cell may be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or a HEK293 cell).

[0145] As used herein, the terms “effective amount” and “therapeutically effective amount” refer to an amount of a polypeptide, nucleic acid, or vector described herein or a pharmaceutical composition containing a polypeptide, nucleic acid, or vector described herein effective in achieving a desired therapeutic effect in treating a patient having a disease or condition, such as a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis or a myelofibrosis treatment in a transfusion-dependent subject. These terms refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating a subject having a cytopenia associated with myelofibrosis in a transfusion-dependent subject, it is an amount of the composition, polypeptide, nucleic acid, or vector sufficient to achieve a treatment response as compared to the response obtained without administration of the composition, polypeptide, nucleic acid, or vector. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition, polypeptide, nucleic acid, or vector of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response. Preferably, the therapeutically effective amount of the polypeptide, nucleic acid, or vector avoids adverse side effects.

[0146] As used herein, the term “pharmaceutical composition” refers to a medicinal or pharmaceutical formulation that includes an active ingredient as well as excipients and diluents to enable the active ingredient suitable for the method of administration. The pharmaceutical composition of the present invention includes pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in tablet or capsule form for oral administration or in aqueous form for intravenous or subcutaneous administration.

[0147] As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier or excipient must provide adequate pharmaceutical stability to a polypeptide described herein (e.g., an ActRII signaling inhibitor, such as an ActRII ligand trap including an extracellular ActRIIA variant), the nucleic acid molecule(s) encoding the polypeptide, or a vector containing such nucleic acid molecule(s). The nature of the carrier or excipient differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0148] As used herein, the term “treating and / or preventing” refers to the treatment and / or prevention of a disease or condition, e.g., a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis or myelofibrosis treatment in a transfusion-dependent subject, using methods and compositions of the invention. Generally, treating a disease or condition, e.g., a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with a myelofibrosis or myelofibrosis treatment in a transfusion-dependent subject, occurs after a subject has developed the disease or condition. Preventing a disease or condition, e.g., a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis or myelofibrosis treatment, refers to steps or procedures taken when a subject is at risk of developing the disease or condition. The subject may show signs or mild symptoms that are judged by a physician to be indications or risk factors for developing the disease or condition, have another disease or condition associated with development of the disease or condition, be undergoing treatment that may cause the disease or condition, or have a family history or genetic predisposition of developing the disease or condition, but has not yet developed the disease or condition.

[0149] As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0150] As used herein, the term “subject” refers to a mammal, e.g., preferably a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., a cynomolgus monkey), mice, dogs, cats, horses, and cows, etc.

[0151] DESCRIPTION OF THE DRAWINGS

[0152] FIG. 1 is a series of graphs showing baseline biomarkers of erythropoiesis in the participants in the Phase 2 study. EPO=erythropoietin, LLN=lower limit of normal, sTfR=soluble transferrin receptor, ULN=upper limit of normal, numbers above the bars are the median value plotted.

[0153] FIGS. 2A-2B are a series of graphs showing observed changes in markers of erythropoiesis in non-TD participants. Abbreviations: g=grams, dl_=deciliter; Hgb=hemoglobin; Non-TD=non-transfusion dependent; sTfR=soluble transferrin receptor; SEM=standard error of the mean. Values for Hgb were censored within 14 days following a transfusion unless they were pre-transfusion values. FIG. 2A shows the mean change in Hgb with data pooled across dose cohorts within the monotherapy and combination arms. FIG. 2B shows the change in Hgb and reticulocytes averaged over 12 weeks and in sTfR over 8 weeks. Given small sample sizes, non-TD participants were pooled across both monotherapy and combination treatment arms at each dose level. Participants were only included in the analysis if they had at least 12 weeks of postbaseline data for reticulocytes and Hgb or 8 weeks for sTfR, leading to different n’s across analytes.

[0154] FIGS. 3A-3B are a series of graphs showing maximum mean change in Hgb observed over 12 weeks in non-TD participants. FIG. 3A shows changes in hemoglobin for participants in the monotherapy (ActRIIA / B-hFc) arm and FIG. 3B shows changes in hemoglobin for participants in the combination therapy (ActRIIA / B-hFc + ruxolitinib) arm.

[0155] FIGS. 4A-4B are a series of graphs showing maximum percent reduction in transfusion burden observed over 12 weeks in participants who received > 3 RBC units / 12 weeks at Baseline. FIG. 4A shows changes in percent RBC units / 12 weeks for participants in the monotherapy (ActRIIA / B-hFc) arm and FIG. 4B shows changes in percent RBC units / 12 weeks for participants in the combination therapy (ActRIIA / B-hFc + ruxolitinib) arm.

[0156] FIGS. 5A-5B are a series of graphs showing mean platelet count observed over time. FIG. 5A shows platelet count for participants in the monotherapy (ActRIIA / B-hFc) arm and FIG. 5B shows platelet count for participants in the combination therapy (ActRIIA / B-hFc + ruxolitinib) arm.

[0157] FIG. 6 is a series of graphs showing data for a participant who achieved trifactor (anemia, spleen, and symptom) improvements.

[0158] FIG. 7 is a series of graphs showing change in markers of erythropoiesis averaged over 12 weeks in Part 1 dose escalation for non-TD participants. Hgb = hemoglobin, sTfR = soluble transferrin receptor. Given small sample sizes, non-TD participants were pooled across both monotherapy and combination treatment arms at each dose level. Participants were only included in the analysis if they had at least 12 weeks of post-baseline data for reticulocytes and Hgb or 8 weeks for sTfR, leading to different sample size across analytes.

[0159] FIGS. 8A-8B show hemoglobin responses observed with ActRIIA / B-hFc treatment. FIG. 8A shows responses observed with monotherapy and FIG. 8B shows responses observed with combination therapy.

[0160] FIGS. 9A-9B show observed decreases in transfusion burden for participants who received > 3 RBC units / 12 weeks at baseline. FIG. 9A shows responses observed with monotherapy and FIG. 9B shows responses observed with combination therapy.

[0161] FIGS. 10A-10B show changes in platelet count. FIG. 10A shows changes observed with monotherapy and FIG. 10B shows changes observed with combination therapy. *Data shown are for the first 24 weeks of treatment in participants with baseline platelets < 150 x109 / L and at least 12 Weeks post-baseline assessments.

[0162] FIGS. 11A-11B show changes in spleen volume from baseline to Week 24. FIG. 11A shows changes observed with monotherapy and FIG. 11 B shows changes observed with combination therapy. FIGS. 12A-12B show changes in symptom score from baseline to Week 24. FIG. 12A shows changes observed with monotherapy and FIG. 12B shows changes observed with combination therapy.

[0163] FIGS. 13A-13B show two case studies - one from the monotherapy arm and one from the combination therapy arm. FIG. 13A is a case study from a participant in the monotherapy arm and FIG. 13B is a case study from a participant in the combination therapy arm. Post-PV MF = post polycythemia vera multiple fibrosis; MF =multiple fibrosis; ULN = upper limit of normal; LLN = lower limit of normal.

[0164] FIGS. 14A-14B show hemoglobin responses observed with ActRIIA / B-hFc treatment. FIG. 14A shows responses observed with monotherapy and FIG. 14B shows responses observed with combination therapy. BL Hgb = baseline hemoglobin; Max Hgb = maximum hemoglobin; ‘Denotes Non-TD participants who received > 3 RBC U / 12 weeks at baseline (TD3).

[0165] FIGS. 15A-15B show observed decreases in transfusion burden for participants who received > 3 RBC units / 12 weeks at baseline (TD3 participants). FIG. 15A shows responses observed with monotherapy and FIG. 15B shows responses observed with combination therapy. BL RBC U = baseline RBC U / 12 weeks; Min RBC U = minimum post-baseline RBC U / 12 weeks; RBC = red blood cells; ‘Denotes TD3 participants who also qualified as transfusion dependent (TD) based on IWG 2013 criteria.

[0166] FIGS. 16A-16B show changes in platelet count over time. FIG. 16A shows changes observed with monotherapy and FIG. 16B shows changes observed with combination therapy. The numbers along the X-axis denote the number of participants in each group with platelet values recorded at each timepoint.

[0167] FIGS. 17A-17B show percent change in spleen volume from baseline to Week 24. FIG. 17A shows changes observed with monotherapy and FIG. 17B shows changes observed with combination therapy. BL = Baseline spleen volume; Rux = ruxolitinib; Wk = week.

[0168] FIGS. 18A-18B show changes in symptom score from baseline to Week 24. FIG. 18A shows changes observed with monotherapy and FIG. 18B shows changes observed with combination therapy. BL = Baseline total score; MF-SAF-TSS = Myelofibrosis symptom assessment form total symptom score; Rux = ruxolitinib; Time on rux = duration of prior ruxolitinib treatment as of the informed consent date; wks = weeks.

[0169] FIGS. 19A-19B show the percent of participants with a > 25% or > 50% reduction in an individual MAF-SAF-TSS item at Week 24. FIG. 19A shows reductions observed with monotherapy and FIG. 19B shows reductions observed with combination therapy.

[0170] DETAILED DESCRIPTION OF THE INVENTION

[0171] The invention features ActRII signaling inhibitors for use in methods of treating myelofibrosis or a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis in a transfusion-dependent subject. The ActRII signaling inhibitor can be administered in combination with a cytopenia-associated myelofibrosis treatment. The ActRII signaling inhibitors described herein can also be used to treat a transfusion-dependent subject receiving treatment with a cytopenia-associated myelofibrosis treatment (e.g., receiving treatment for myelofibrosis or another disease or condition, such as polycythemia vera, steroid-refractory graft-versus-host disease, or chronic graft-versus-host disease). The ActRII signaling inhibitor can be an antibody that binds to an ActRII ligand, an anti-ActRII antibody, or an ActRII ligand trap (e.g., an ActRIIA ligand trap, ActRIIB ligand trap, or ActRII chimera ligand trap, such as an ActRIIA ligand trap including an extracellular ActRIIA variant), and exemplary cytopenia-associated myelofibrosis treatments include ruxolitinib (JAKAFIO / JAKAVI®), fedratinib (INREBIC®), pacritinib (VONJO™), and imetelstat. In some embodiments, an ActRII ligand trap includes an extracellular ActRI I variant fused to a moiety (e.g., Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin). The ActRII signaling inhibitors described herein, such as an ActRIIA ligand trap including an extracellular ActRIIA variant described herein, can treat myelofibrosis, a myelofibrosis-associated cytopenia, or a subject receiving a cytopenia-associated myelofibrosis treatment by increasing hemoglobin, reducing transfusion burden, maintaining or increasing platelet count, improving splenomegaly, and improving constitutional symptoms.

[0172] ActRII signaling

[0173] Activin type II receptors are single transmembrane domain receptors that modulate signals for ligands in the transforming growth factor p (TGF-p) superfamily. Ligands in the TGF-p superfamily are involved in a host of physiological processes, such as muscle growth, vascular growth, cell differentiation, homeostasis, and osteogenesis. Examples of ligands in the TGF-p superfamily include, e.g., activin A, activin B, inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin, and GDF11), and bone morphogenetic proteins (BMPs) (e.g., BMP9).

[0174] TGF-p signaling pathways regulate hematopoiesis, with signaling pathways involving activins preventing the differentiation of red blood cell, platelet, and neutrophil progenitor cells in order to maintain progenitor cells in a quiescent state and signaling pathways involving BMPs promoting differentiation of progenitor cells. Homeostasis of this process is essential to ensure that all cell types, including red cells, white cells, and platelets, are properly replenished in the blood.

[0175] The present invention is based, in part, on the discovery that administration of a polypeptide including an ActRIIA variant described herein (an ActRIIA variant-Fc polypeptide) to human subjects having myelofibrosis and anemia alone or in combination with ruxolitinib reduced transfusion burden in subjects receiving three or more RBC units per 12 weeks at baseline, even in subjects with more severe transfusion burden. Increases in markers of erythropoiesis were also observed, as well as increases in hemoglobin, maintenance or improvement in platelet count, reduction in spleen size, and a reduction in disease symptoms. Accordingly, these data suggest that ActRII signaling inhibitors, such as an ActRIIA variant described herein, could be used be used to treat transfusion-dependent subjects having myelofibrosis, a cytopenia (e.g., anemia) associated with myelofibrosis, and / or receiving treatment with a cytopenia-associated myelofibrosis treatment (e.g., subjects having a cytopenia associated with ruxolitinib).

[0176] ActRII signaling inhibitors

[0177] ActRII signaling inhibitors are agents that reduce or prevent the interaction of ActRII ligands with ActRIIA and / or ActRIIB, by either binding to the ligand or to the receptor. ActRII signaling inhibitors for use in the methods described herein provided herein below.

[0178] In some embodiments, the ActRII signaling inhibitor is an activin A antibody or an antigen binding fragment thereof. In some embodiments, the activin A antibody is garetosmab (also known as REGN- 2477). Additional activin A antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2015017576, WO2013074557, W02008031061 , and WO2023147107; US Patent Application No. US20150359850; and US Patent Nos. 9,718,881 , 10,526,403, 8,309,082, 8,753,627, and 10,100,109, each of which is incorporated herein by reference.

[0179] In some embodiments, the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) and a light chain variable region (LCVR) listed in Table 1 (e.g., an HCVR and an LCVR from the same row of Table 1). In some embodiments, the activin A antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , such as any one of SEQ ID NOs: 138, 140, 142, 143, 144, 146, 148, 150, 151 , 172, 174, 834, 836, 838, 840, 842, 843, 845, 847, 848, 850, 852, 854, 856, 858, 859, 861 , 863, and 865, and a LCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 , such as any one of SEQ ID NOs: 139, 141 , 145, 147, 149, 173, 175, 835, 837, 839, 841 , 844, 846, 849, 851 , 853, 855, 857, 860,862, and 864. In some embodiments, the activin A antibody or an antigen binding fragment thereof, apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 1 . In some embodiments, the activin A antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 1 . In some embodiments, the activin A antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 1 .

[0180] Table 1. Exemplary HCVR and LCVR sequences of activin A antibodies

[0181] In some embodiments, the activin A antibody or an antigen-binding fragment thereof, has the CDR sequences described in Table 2 (i.e., a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3). In some embodiments, the activin A antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 2, such as any one of SEQ ID NOs: 155, 161 , 179, 185, 869, 875, 884, 903, 914, 925, and 931 ; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 2, such as any one of SEQ ID NOs: 156, 162, 180, 186, 870, 936, 885, 894, 898, 904, 909, 915, 926, and 932; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR3 sequence in Table 2, such as any one of SEQ ID NOs: 157, 163, 181 , 187, 871 , 876, 937, 879, 886, 889, 895, 899, 905, 910, 916, 921 , 927, and 933; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a heavy chain variable CDR1 sequence in Table 2, such as any one of SEQ ID NOs: 152, 158, 176, 182, 866, 872, 877, 938, 880, 882, 887, 891 , 896, 900, 906, 911 , 918, 922, 928, and 934; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 2, such as any one of SEQ ID NOs: 153, 159, 177, 183, 867, 873, 878, 881 , 883, 888, 890, 892, 897, 901 , 907, 912, 917, 919, 923, 929, and 935; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR3 sequence in Table 2, such as any one of SEQ ID NOs: 154, 160, 178, 184, 868, 874, 893, 902, 908, 913, 920, 924, and 930. In some embodiments, the activin A antibody or antigen binding fragment thereof includes a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 2. Table 2. Exemplary CDR sequences of activin A antibodies

[0182] In some embodiments, the activin A antibody or an antigen-binding fragment thereof, has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 3. In some embodiments, the activin A antibody or an antigen binding fragment thereof, has a heavy chain and light chain sequence from the same row of Table 3. In some embodiments, the heavy chain and light chain have the sequence of SEQ ID NOs: 939 and 940; 941 and 942; 943 and 944; 1026 and 945; 946 and 940; 947 and 948; 949 and 950; 951 and 950; 952 and 953; 954 and 955; 956 and 957; 958 and 959; 960 and 961 ; 962 and 961 ; 963 and 964; 965 and 966; 967 and 968; or 969 and 970 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%,

[0183] 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the second SEQ ID NO: in each pair). Table 3. Exemplary heavy and light chain sequences of activin A antibodies

[0184] In some embodiments, the ActRII signaling inhibitor is a myostatin antibody or an antigen binding fragment thereof. In some embodiments, the myostatin antibody is domagrozumab (also known as PF- 06252616), landogrozumab (also known as LY2495655), trevogrumab (also known as REGN-1033), or apitegromab (SRK-015). Additional myostatin antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. W02007047112, W02007044411 , W02006116269, WO2012024242, WO2016073853, WO2013186719, W02009058346, WO2011150008, WO2016168613, W02007024535, WO2016098357, WO2022093724, WO2017049011 , and WO2017120523, US Patent Application Nos. US20070178095 and US20210246198; and US Patent Nos. 10,000,560, 10,738,111 , 7,632,499, 8,066,995, 7,635,760, 7,745,583, 7,807,159, 8,999,343, 10,307,480, 8,992,913, 9,751 ,937, 9,409,981 , 9,850,301 , 8,840,894, 9,890,212, 9,260,515, 10,934,349, 8,871 ,209, 10,400,036, 7,888,486, and 8,372,625, each of which is incorporated herein by reference.

[0185] In some embodiments, the myostatin antibody or an antigen binding fragment thereof has a HCVR and a LCVR listed in Table 4 (e.g., an HCVR and an LCVR from the same row of Table 4). In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4, such as any one of SEQ ID NOs: 164, 188, 201 , 204- 210, 222-228, 234, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 298, 306, 308, 310, 312, 314, 316, 318, 320, 356, 371 , 373, 387, 389, 391 , 405, 407, 409, 411 , 413, 415, 417, 419, 421-423, 425, 427, 429, 431 , 433, 435, 437, 439, 441 , 444, 446, 448-476, 971 , 973, 975, 977, 979, 981 , 983, 985, 987, 989, 991 , 993, 995, 997, 999, 10001 , and 10003, and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4, such as any one of SEQ ID NOs: 165, 189, 202, 203, 221 , 229-233, 251 , 253, 255,

[0186] 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 299, 307, 309, 311 , 313, 315, 317, 319, 321 , 358, 372, 374,

[0187] 388, 390, 392, 406, 408, 410, 412, 414, 416, 418, 420, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,

[0188] 443, 445, 447, 477-486, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000,

[0189] 1002, and 1004. In some embodiments, the myostatin antibody or an antigen binding fragment thereof, apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 4. In some embodiments, the myostatin antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 4. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 4. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes an HCVR sequence of any one of SEQ ID NOs: 448-476 and an LCVR sequence of any one of SEQ ID NOs: 477-486. Table 4. Exemplary HCVR and LCVR sequences of myostatin antibodies

[0190]

[0191] In some embodiments, the myostatin antibody or an antigen-binding fragment thereof, has the CDR sequences described in Table 5, 6, or 7 (i.e. , a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3). In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 169, 193, 198, 238, 241 , 303, 325, 330, 362, 378, 384, 396, 402, 826, 490, 493, 343-346, 1007, 1013, 1017, 1020, 1023; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 170, 194, 199, 239, 304, 326, 331 , 363, 379, 385, 397, 403, 827, 491 , 347-349, 1008, 1014, 1021 ; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR3 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 171 , 195, 200, 240, 245, 249, 305, 327, 364, 380, 386, 398, 404, 828, 492, 350-355, 1009, 1015; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a heavy chain variable CDR1 sequence in Table 5 or Table 6, such as any one of SEQ ID NOs: 166, 190 196, 235, 242, 246, 300, 322, 328, 359, 366, 375, 381 , 393, 399, 823, 487, 332-334, 1005, 1010, 1018; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 5 or Table 6, such as any one of SEQ ID NOs: 167, 191 , 197, 236, 243, 247, 301 , 323, 329, 360, 365, 376, 382, 394, 400, 824, 488, 335, 336, 1011 , 1016, 1019, 1022; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR3 sequence in Table 5 or Table 6, such as any one of SEQ ID NOs: 168, 192, 237, 244, 248, 302, 324, 361 , 377, 383, 395, 401 , 825, 489, 337-342, 1006, 1012. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 5.

[0192] Table 5. Exemplary CDR sequences of myostatin antibodies

[0193] Table 6. Exemplary heavy chain CDR sequences of myostatin antibodies

[0194] Table 7. Exemplary light chain CDR sequences of myostatin antibodies

[0195] In some embodiments, the myostatin antibody or an antigen-binding fragment thereof, has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 8. In some embodiments, the myostatin antibody or an antigen binding fragment thereof, has a heavy chain and light chain sequence from the same row of Table 8. In some embodiments, the heavy chain and light chain have the sequence of SEQ ID NOs: 274 and 275; 276 and 277; 278 and 279; 280 and 281 ; 282 and 283; 284 and 285; 286 and 287; 288 and 289; 290 and 291 ; 292 and 293; 294 and 295; 296 and 297; 367 and 368; 69 and 370, or 1024 and 1025 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the second SEQ ID NO: in each pair).

[0196] Table 8. Exemplary heavy and light chain sequences of myostatin antibodies

[0197] In some embodiments, the myostatin antibody is a bi-specific antibody that also binds to activin A. Exemplary bi-specific myostatin antibodies that may be used in the methods described herein include those described in US Patent Nos. 9,718,881 , 10,526,403, 10,400,036 and 8,871 ,209, the disclosures of which are incorporated herein by reference. In some embodiments, the bi-specific antibody includes an activin A HCVR and LCVR from Table 1 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , such as any one of SEQ ID NOs: 138, 140, 142, 143, 144, 146, 148, 150, 151 , 172, and 174, and a LCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 , such as any one of SEQ ID NOs: 139, 141 , 145, 147, 149, 173, and 175) and a myostatin HCVR and LCVR from Table 4 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4, such as any one of SEQ ID NOs: 164, 188, 201 , 204-210, 222- 228, 234, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 298, 306, 308, 310, 312, 314, 316, 318, 320, 356, 371 , 373, 387, 389, 391 , 405, 407, 409, 411 , 413, 415, 417, 419, 421-423, 425, 427, 429, 431 , 433, 435, 437, 439, 441 , 444, 446, and 448-476, and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4, such as any one of SEQ ID NOs: 165, 189, 202, 203, 221 , 229-233, 251 , 253, 255,

[0198] 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 299, 307, 309, 311 , 313, 315, 317, 319, 321 , 358, 372, 374,

[0199] 388, 390, 392, 406, 408, 410, 412, 414, 416, 418, 420, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,

[0200] 443, 445, 447, and 477-486). In some embodiments, the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 2 (e.g., an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 2) and a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 5 (e.g., a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 5). In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 138 and LCVR of SEQ ID NO: 139 and a myostatin HCVR of SEQ ID NO: 164 and LCVR of SEQ ID NO: 165. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 138 and LCVR of SEQ ID NO: 139 and a myostatin HCVR of SEQ ID NO: 387 and LCVR of SEQ ID NO: 388. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 138 and LCVR of SEQ ID NO: 139 and a myostatin HCVR of SEQ ID NO: 391 and LCVR of SEQ ID NO: 392. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 144 and LCVR of SEQ ID NO: 145 and a myostatin HCVR of SEQ ID NO: 164 and LCVR of SEQ ID NO: 165. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 144 and LCVR of SEQ ID NO: 145 and a myostatin HCVR of SEQ ID NO: 387 and LCVR of SEQ ID NO: 388. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 144 and LCVR of SEQ ID NO: 145 and a myostatin HCVR of SEQ ID NO: 391 and LCVR of SEQ ID NO: 392. In some embodiments, the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 152-157 and a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 166-171. In some embodiments, the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 158-163 and a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 166-171.

[0201] In some embodiments, the ActRII signaling inhibitor is an activin B antibody or an antigen binding fragment thereof. Activin B antibodies that may be used in the methods described herein include those described in US Patent No. 8,383,351 , which is incorporated herein by reference. In some embodiments, the activin B antibody or an antigen binding fragment thereof has a HCVR including three CDRs from the HCVR sequence of SEQ ID NO: 494 and a LCVR including three CDRs from the LCVR sequence of SEQ ID NO: 495. In some embodiments, the activin B antibody or an antigen binding fragment thereof has a HCVR having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 494. In some embodiments, the activin B antibody or an antigen binding fragment thereof has a LCVR having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 495.

[0202] MKCSWIMFFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMYWVK QRPGQGLEWIGMIHPNSGSTNYNGKFKTGATLTVDKSSSTVYMQLSSLTSEDSAVYYC ARWGYGGNYDYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSL (SEQ ID NO: 494)

[0203] MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQ QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISTMEAEDAVTYYCHQYHRSP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK (SEQ ID NO: 495)

[0204] In some embodiments, the ActRII signaling inhibitor is a GDF-11 antibody or an antigen binding fragment thereof. In some embodiments, the ActRII signaling inhibitor is ActRII antibody or an antigen binding fragment thereof. There exist two types of activin type II receptors: ActRIIA and ActRIIB. In some embodiments, the ActRII antibody is an ActRIIA antibody or an antigen binding fragment thereof. In some embodiments, the ActRII antibody is an ActRIIB antibody or an antigen binding fragment thereof. In some embodiments, the ActRII antibody or an antigen binding fragment thereof binds to both ActRIIA and ActRIIB. In some embodiments, the ActRII antibody is bimagrumab (also known as BYM338), CSJ089, CQI876, or CDD861 (described in Morvan et al., PNAS 114:12448-12453 (2017)). Additional ActRII antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2010125003, WO2012064771 , WO2017156488, WO2013063536, WO2018175460, WO2021044287, WO2013188448, and WO2020243448; US Patent Application No. US20180066061 , US20180230221 , US20180111991 , US20200181271 , US20210309749, and US20160200818; and US Patent Nos. 9,453,080, 10,266,598, 10,981 ,999, 10,307,455, 11 ,000,565, 10,982,000, 9,969,806, 9,365,651 , 8,388,968, 8,551 ,482, 9,493,556, 8,765,385, and 9,624,301 , each of which is incorporated herein by reference.

[0205] In some embodiments, the ActRII antibody or an antigen binding fragment thereof has a HCVR and a LCVR listed in Table 9 (e.g., an HCVR and an LCVR from the same row of Table 9). In some embodiments, the ActRII antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 9, such as any one of SEQ ID NOs: 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 583, 591 , 593, 595-598, 600, 602, 603, 605, 606, 608, 610-614, 687, 689, 692, 695, and 697, and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 9, such as any one of SEQ ID NOs: 513, 515, 517, 519, 521 , 523, 525, 527, 529, 531 , 533, 535, 537, 539, 584, 592, 594, 601 , 604, 607, 609, 615, 688, 690, 691 , 693, 694, 696, and 698. In some embodiments, the ActRII antibody or an antigen binding fragment thereof, apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 9. In some embodiments, the ActRII antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 9. In some embodiments, the ActRII antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 9.

[0206] Table 9. Exemplary HCVR and LCVR sequences of ActRII antibodies

[0207] In some embodiments, the ActRII antibody or an antigen-binding fragment thereof, has the CDR sequences described in Table 10 (i.e., a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3). In some embodiments, the ActRII antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 10, such as any one of SEQ ID NOs: 499, 505, 543, 580, 588, 619, 625, 633, 640, 648, 654, 663, 684, 702, 705, 711 , 714, 720, and 726; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 10, such as any one of SEQ ID NOs: 500, 506, 544, 581 , 589, 620, 626, 634, 641 , 649, 655, 664, 685, 703, 706, 712, 715, 721 , and 727; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR3 sequence in Table 10, such as any one of SEQ ID NOs: 501 , 507, 545, 547, 548, 549, 582, 590, 621 , 627, 635, 635, 642, 650, 656, 665, 686, 704, 707, 713, 716, 722, and 728; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91 %, 92%, 93%,

[0208] 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a heavy chain variable CDR1 sequence in Table 10, such as any one of SEQ ID NOs: 496, 502, 540, 577, 585, 616, 622, 629, 630, 638, 644, 651 , 659, 660, 669-672, 679-681 , 699, 708, 717, and 723; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 10, such as any one of SEQ ID NOs: 497, 503, 541 , 546, 550-556, 578, 586, 617, 623, 628, 631 , 637, 643, 646, 652, 658, 661 , 666, 667, 668, 676, 677, 678, 682, 700, 709, 718, and 724; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR3 sequence in Table 10, such as any one of SEQ ID NOs: 498, 504, 542, 579, 587, 618, 624, 632, 636, 639, 647, 653, 657, 662, 673, 674, 675, 683, 701 , 710, 719, and 725. In some embodiments, the ActRII antibody or antigen binding fragment thereof includes a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 10.

[0209] Table 10. Exemplary CDR sequences of ActRII antibodies

[0210] In some embodiments, the ActRII antibody or an antigen-binding fragment thereof, has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 11 . In some embodiments, the ActRII antibody or an antigen binding fragment thereof, has a heavy chain and light chain sequence from the same row of Table 1 1 . In some embodiments, the heavy chain and light chain have the sequence of SEQ ID NOs: 508 and 509; 510 and 511 ; 557 and 558; 559 and 560; 561 and 562; 563 and 564; 565 and 566; 567 and 568; 569 and 570; 571 and 572; 573 and 574; or 575 and 576 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID

[0211] NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the second SEQ ID NO: in each pair). Table 11 . Exemplary heavy and light chain sequences of ActRII antibodies

[0212] In some embodiments, the ActRII signaling inhibitor is an ActRII ligand trap. ActRII ligand traps are polypeptides that contain an extracellular portion of ActRIIA and / or ActRIIB that are capable of binding to one or more ActRII ligands (e.g., activin A, activin B, myostatin, or GDF11). The extracellular portion of ActRIIA and / or ActRIIB may be fused to a moiety (e.g., an Fc domain, an Fc domain monomer, an albumin-binding peptide, a fibronectin domain, or a human serum albumin) by way of a linker. ActRII ligand traps can reduce or inhibit the binding of ActRII ligands to endogenous activin type II receptors, thereby reducing ActRII signaling. As the ActRII ligand traps contain the extracellular portion of the receptor, they will be soluble and able to bind to and sequester ligands (e.g., activins A and B, myostatin, GDF11) without activating intracellular signaling pathways.

[0213] In some embodiments, the ActRII ligand trap is an ActRIIA ligand trap. The ActRIIA ligand trap may contain an extracellular portion of wild-type ActRIIA (e.g., human or murine ActRIIA) or may contain an extracellular portion of wild-type ActRIIA that contains one or more amino acid substitutions relative to the wild-type human extracellular ActRIIA. The wild-type amino acid sequence of the extracellular portion of human ActRIIA is shown below. Human ActRIIA, extracellular portion (SEQ ID NO: 73):

[0214] GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQG CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS

[0215] An ActRIIA ligand trap may contain the sequence of SEQ ID NO: 73 or a variant thereof that contains one or more amino acid substitutions. In some embodiments, the ActRIIA ligand trap contains a portion of SEQ ID NO: 73 (e.g., a contiguous portion that is shortened by the removal of amino acids from the N-terminus, C-terminus, or both) or a variant thereof that contains one or more amino acid substitutions. In some embodiments, the ActRIIA ligand trap contains the sequence of SEQ ID NO: 73 or a portion thereof with additional amino acids at the C-terminus from the wild-type sequence of ActRIIA (SEQ ID NO: 75). An exemplary sequence of a portion of wild-type ActRIIA protein that is shortened at the N-terminus and includes additional amino acids from SEQ ID NO: 75 at the C-terminus that can be included in an ActRIIA ligand trap is provided below:

[0216] ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP (SEQ ID NO: 729)

[0217] Studies have shown that BMP9 binds ActRIIB with about 300-fold higher binding affinity than ActRIIA (see, e.g., Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIA-Fc is known to have a longer half-life compared to ActRIIB-Fc. Described herein below are ActRIIA ligand traps containing extracellular ActRIIA variants that are constructed by introducing amino acid residues of ActRIIB to ActRIIA, with the goal of imparting physiological properties conferred by ActRIIB, while also maintaining beneficial physiological and pharmacokinetic properties of ActRIIA. The optimum peptides promote hematopoiesis (e.g., increase red blood cell count, hemoglobin levels, hematocrit, reticulocytes, platelet levels (e.g., platelet count), and / or neutrophil levels (e.g., neutrophil count)), while retaining low bindingaffinity to BMP9 and longer serum half-life as an Fc fusion protein, for example. The preferred ActRIIA variants also exhibit similar or improved binding to activins and / or myostatin compared to wild-type ActRIIA, which allows them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling. These variants can be used to treat a cytopenia (e.g., anemia, thrombocytopenia, and / or neutropenia) associated with myelofibrosis by increasing hemoglobin levels, hematocrit, red blood cell count (e.g., increasing red blood cell production and / or red cell mass or volume), or erythroid progenitor maturation and / or differentiation (e.g., the maturation and / or differentiation of early-stage or late- (e.g., terminal) stage erythroid progenitors into proerythroblasts, reticulocytes, or red blood cells), reducing the accumulation of red blood cell progenitor cells (e.g., by stimulating progenitor cells to progress to maturation), increasing late-stage precursor (erythroid precursor) maturation (e.g., terminal maturation, such as the maturation of reticulocytes into red blood cells, or the maturation of erythroblasts into reticulocytes and / or red blood cells), recruiting early-stage progenitors into the erythroid lineage, increasing the number of early-stage erythroid precursors and / or progenitors, promoting the progression of erythroid precursors and / or progenitors through erythropoiesis (e.g., progression through the erythropoiesis pathway), increasing proerythroblasts, increasing reticulocytes, increasing platelet levels (e.g., increasing platelet count, megakaryocyte differentiation and / or maturation, megakaryocyte progenitor renewal, and / or platelet production), reducing the accumulation of platelet progenitor cells (e.g., by stimulating progenitor cells to progress to maturation), increasing neutrophil levels (e.g., increasing neutrophil count, e.g., increasing neutrophil production), and / or increasing the differentiation and / or maturation of progenitor cells (e.g., myeloid progenitors, myeloblasts, or myelocytes) into neutrophils. In some embodiments, amino acid substitutions may be introduced to an extracellular ActRIIA variant to reduce or remove the binding affinity of the variant to BMP9.

[0218] ActRIIA ligand traps described herein can include an extracellular ActRIIA variant having at least one amino acid substitution relative to the wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73. Possible amino acid substitutions at 27 different positions may be introduced to an extracellular ActRIIA variant (Table 12). In some embodiments, an extracellular ActRIIA variant may have at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 73). An extracellular ActRIIA variant may have one or more (e.g., 1-27, 1-25, 1-23, 1-21 , 1-19, 1-17, 1-15, 1-13, 1-11 , 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27) amino acid substitutions relative the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 73). In some embodiments, an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of SEQ ID NO: 1) may include amino acid substitutions at all of the 27 positions as listed in Table 12. In some embodiments, an extracellular ActRIIA variant may include amino acid substitutions at a number of positions, e.g., at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 out of the 27 positions, as listed in Table 12.

[0219] Amino acid substitutions can worsen or improve the activity and / or binding affinity of the ActRIIA variants of the invention. To maintain polypeptide function, it is important that the lysine (K) at position Xi? in the sequences shown in Tables 12 and 13 (SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) be retained. Substitutions at that position can lead to a loss of activity. For example, an ActRIIA variant having the sequence: GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYD RTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 85) has reduced activity in vivo, indicating that the substitution of alanine (A) for lysine (K) at X17 is not tolerated. ActRIIA variants of the invention, including variants in Tables 12 and 13 (e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72), therefore, retain amino acid K at position X17.

[0220] The ActRIIA variants of the invention preferably have reduced, weak, or no substantial binding to BMP9. BMP9 binding is reduced in ActRIIA variants (e.g., reduced compared to wild-type ActRIIA) containing the amino acid sequence TEEN (SEQ ID NO: 76) at positions X23, X24, X25, and X26, as well as in variants that maintain the amino acid K at position X24 and have the amino acid sequence TKEN (SEQ ID NO: 77) at positions X23, X24, X25, and X26. The sequences TEEN (SEQ ID NO: 76) and TKEN (SEQ ID NO: 77) can be employed interchangeably in the ActRIIA variants (e.g., the variants in Tables 12 and 13, e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) of the invention to provide reduced BMP9 binding. The ActRIIA variants of the invention may further include a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension can add one or more additional amino acids at the C-terminus (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) to any of the variants shown in Tables 12 and 13 (e.g., SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA. One potential C-terminal extension that can be included in the ActRIIA variants of the invention is amino acid sequence NP. For example, a sequence including the C-terminal extension NP is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C- terminal extension of NP). Another exemplary C-terminal extension that can be included in the ActRIIA variants of the invention is amino acid sequence NPVTPK (SEQ ID NO: 78). For example, a sequence including the C-terminal extension NPVTPK (SEQ ID NO: 78) is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK (SEQ ID NO: 78)).

[0221] Table 12. Amino acid substitutions in an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-5

[0222] In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X3 is E, Xs is R, Xu is D, X12 is K, X13 is R, X16 is K or R, X17 is K, X19 is W, X20 is L, X21 is D, and X22 is I or F. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 , X2 is Y; X4 is L; Xs is E; X9 is E; X14 is L; Xis is K; X23 is T; X25 is E; X26 is N; and X27 is Q. These substitutions in SEQ ID NO: 1 can also be made in SEQ ID NOs: 2-5. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 , Xi is F or Y; X2 is Y; X4 is L; X5 is D or E; X7 is P or R; Xs is E; X9 is E; X10 is K or Q; X14 is L; X15 is F or Y; X16 is K or R; Xis is K; X22 is I or F; X23 is T; X24 is K or E; X25 is E; X26 is N; and X27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 , Xi is F or Y; X2 is Y; X3 is E; X4 is L; X5 is D or E; Xs is R; X7 is P or R; Xs is E; X9 is E; X10 is K or Q; Xu is D; X12 is K; X13 is R; X14 is L; X15 is F or Y; X16 is K or R; X17 is K; Xis is K; X19 is W; X20 is L; X21 is D; X22 is I or F; X23 is T; X24 is K or E; X25 is E; X26 is N; and X27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X17 is K. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NOs: 1-3, X17 is K, X23 is T, X24 is E, X25 is E, and X26 is N. In some embodiments of the extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-5, X17 is K, X23 is T, X24 is K, X25 is E, and X26 is N. In some embodiments, an ActRIIA ligand trap described herein includes an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 6-72 (Table 13).

[0223] Table 13. Extracellular ActRIIA variants having the sequences of SEQ ID NOs: 6-72

[0224] In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) has amino acid K at position X17. Altering the amino acid at position X17 can result in reduced activity. For example, an ActRIIA variant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYD RTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 85) has reduced activity in vivo, indicating that the substitution of A for K at X17 is not tolerated.

[0225] In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) with the sequence TEEN (SEQ ID NO: 76) at positions X23, X24, X25, and X26 can have a substitution of the amino acid K for the amino acid E at position X24. In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) with the sequence TKEN (SEQ ID NO: 77) at positions X23, X24, X25, and X26 can have a substitution of the amino acid E for the amino acid K at position X24. ActRIIA variants having the sequence TEEN (SEQ ID NO: 76) or TKEN (SEQ ID NO: 77) at positions X23, X24, X25, and X26 have reduced or weak binding to BMP9 (e.g., reduced binding to BMP9 compared to BMP9 binding of wild-type ActRIIA).

[0226] In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) may further include a C-terminal extension (e.g., one more additional amino acids at the C-terminus). The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA. In some embodiments, the C-terminal extension is amino acid sequence NP. For example, a sequence including the C-terminal extension NP is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). In some embodiments, the C-terminal extension is amino acid sequence NPVTPK (SEQ ID NO: 78). For example, a sequence including the C-terminal extension NPVTPK (SEQ ID NO: 78) is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK (SEQ ID NO: 78)). The C-terminal extension can add one or more amino acids at the C- terminus (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids).

[0227] In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant may further include a moiety (e.g., Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin), which may be fused to the N- or C-terminus (e.g., C- terminus) of the extracellular ActRIIA variant by way of a linker or other covalent bonds. A polypeptide including an extracellular ActRIIA variant fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.

[0228] Furthermore, in some embodiments, an ActRIIA ligand trap described herein (e.g., an ActRIIA variant-Fc fusion protein) has a serum half-life of at least 7 days in humans. The ActRIIA ligand trap may bind to activin A with a KD of 10 pM or higher. In some embodiments, the ActRIIA ligand trap does not bind to BMP9 or activin A. In some embodiments, the ActRIIA ligand trap binds to activin A, activin B, and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., reduced BMP9 binding compared to BMP9 binding of wild-type ActRIIA). In some embodiments, the ActRIIA ligand trap that has reduced or weak binding to BMP9 has the sequence TEEN (SEQ ID NO: 76) or TKEN (SEQ ID NO: 77) at positions X23, X24, X25, and X26. In some embodiments, the ActRIIA ligand trap does not substantially bind to human BMP9.

[0229] In some embodiments, the ActRIIA ligand trap may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM). In some embodiments, the ActRIIA ligand trap may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM) The ActRIIA ligand trap may also bind to growth and differentiation factor 1 1 (GDF-11) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher).

[0230] In some embodiments, the ActRIIA ligand trap is sotatercept (also known as ACE-011). Additional ActRIIA ligand traps that may be used in the methods described herein are described in International Patent Application Publication No. W02007062188 and US Patent Nos. 7,709,605, 9,138,459, 7,612,041 , 8,067,360, 8,629,109, 9,572,865, 9,163,075, 10,071 ,135, and 7,951 ,771 , each of which is incorporated herein by reference.

[0231] In some embodiments, the ActRII ligand trap is an ActRIIB ligand trap. The ActRIIB ligand trap may contain an extracellular portion of wild-type ActRIIB (e.g., human or murine ActRIIB) or may contain an extracellular portion of wild-type ActRIIB that contains one or more amino acid substitutions relative to the wild-type human extracellular ActRIIB. The wild-type amino acid sequence of the extracellular portion of human ActRIIB is shown below.

[0232] Human ActRIIB, extracellular portion (SEQ ID NO: 74):

[0233] GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKG CWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAP T

[0234] An ActRIIB ligand trap may contain the sequence of SEQ ID NO: 74 or a variant thereof that contains one or more amino acid substitutions. In some embodiments, the ActRIIB ligand trap contains a portion of SEQ ID NO: 74 (e.g., a contiguous portion that is shortened by the removal of amino acids from the N-terminus, C-terminus, or both) or a variant thereof that contains one or more amino acid substitutions. For example, the ActRIIB ligand trap can include the sequence of SEQ ID NO: 74 with an L60D substitution. In another example, the ActRIIB ligand trap can include the sequence of SEQ ID NO: 74 with a substitution at position E9 (e.g., an E9W, E9A, E9F, E9Q, E9V, E9I, E9L, E9M, E9K, E9H, or E9Y substitution), an S25T substitution, and / or an R45A substitution. In some embodiments, the ActRIIB ligand trap is BIIB110 (previously known as ALG-801), ALG-802, luspatercept (REBLOZYL®, also known as ACE-536), Ramatercept (also known as ACE-031), or ACE-2494. Additional ActRIIB ligand traps that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2010062383, WO2015192127, WO2019140283, and WO2021189010; US Patent Application Publication Nos. US20110250198 and US20200407415; and US Patent Nos. 10,913,782, 8,058,229, 8,216,997, 8,703,927, 9,439,945, 9,932,379, 10,131 ,700, 10,689,427, 10,889,626, 10,829,532, 10,829,533, 8,361 ,957, 9,505,813, 10,377,996, 9,617,319, 8,710,016, 7,709,605, 8,252,900, 7,842,663, 8,343,933, 9,399,669, 10,259,861 , 8,138,142, 8,178,488, 8,293,881 , 9,181 ,533, 9,745,559,

[0235] 10,358,633, 11 ,066,654, 9,610,327, 9,284,364, 8,067,562, 8,614,292, 7,947,646, 8,716,459, 8,501 ,678, 8,999,917, 9,447,165, 9,809,638, 10,407,487, 8,410,043, 9,273,114, and 10,308,704, each of which is incorporated herein by reference.

[0236] In some embodiments, the ActRIIB ligand trap contains an ActRIIB variant having the sequence of SEQ ID NO: 730 shown in Table 14.

[0237] Table 14. Amino acid substitutions in an extracellular ActRIIB variant having a sequence of SEQ ID NO: 730 In some embodiments, the ActRIIB variant has the sequence of any one of SEQ ID NOs: 731-744

[0238] (Table 15). Table 15. Extracellular ActRIIB variants having the sequences of SEQ ID NOs: 731-744

[0239] In some embodiments, the extracellular ActRIIB variant has an N-terminal truncation of 1-7 amino acids (e.g., 1 , 2, 3, 4, 5, 6, or 7 amino acids). An N-terminal truncation can be produced by removing 1-7 amino acids from the N-terminus of an of an ActRIIB variant shown in Tables 14 and 15. The N-terminal truncation can remove amino acids up two to amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE) are retained in the N-terminally truncated ActRIIB variants). Additional ActRIIB variants having an N-terminal truncation are provided below: ETRECIYYNANWELERTNQSGLERCYGDKDKRRHCYASWRNSSGTIELVKKGCWLDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 745)

[0240] ETRECIYYNANWELERTNQSGLERCEGDQDKRLHCYASWRNSSGTIELVKKGCWLDDI NCYDRQECVATKENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 746)

[0241] ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT (SEQ ID NO: 747)

[0242] ETRWCIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 748)

[0243] ETRWCIYYNANWELERTNQTGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 749)

[0244] ETRYCIYYNANWELERTNQTGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDF NCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 750)

[0245] In some embodiments, an ActRIIB ligand trap including an ActRIIB variant may further include a moiety (e.g., Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin), which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIB variant by way of a linker or other covalent bonds. An ActRIIB ligand trap including an extracellular ActRIIB variant fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.

[0246] Furthermore, in some embodiments, an ActRIIB ligand trap described herein has a serum half-life of at least 7 days in humans. The ActRIIB ligand trap may bind to bone morphogenetic protein 9 (BMP9) with a KD of 200 pM or higher. The ActRIIB ligand trap may bind to activin A with a KD of 10 pM or higher. In some embodiments, the ActRIIB ligand trap does not bind to BMP9 or activin A. In some embodiments, the ActRIIB ligand trap binds to activin and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9.

[0247] Additionally, in some embodiments, the ActRIIB ligand trap may bind to human BMP9 with a KD of about 200 pM or higher (e.g., a KD of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or higher, e.g., a KD of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or higher, e.g., a KD of between about 200 pM and about 50 nM). In some embodiments, the ActRIIB ligand trap does not substantially bind to human BMP9. In some embodiments, the ActRIIB ligand trap may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM). In some embodiments, the ActRIIB ligand trap may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM) The ActRIIB ligand trap may also bind to growth and differentiation factor 11 (GDF-11) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher).

[0248] In some embodiments, the ActRII ligand trap is an ActRII chimera ligand trap. The ActRII chimera ligand traps contain portions of extracellular ActRIIA (e.g., human ActRIIA) and extracellular ActRIIB (e.g., human ActRIIB). In some embodiments, the ActRII chimera ligand traps contain an N-terminal portion of extracellular ActRIIB (SEQ ID NO: 74 shown above) joined to a C-terminal portion of extracellular ActRIIA (SEQ ID NO: 73 shown above) such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA). In some embodiments, the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap includes the six amino acids found at the N-terminus of extracellular ActRIIA joined to the fifth amino acid of extracellular ActRIIB. In some embodiments, the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap begins with the first amino acid located at the N-terminus of extracellular ActRIIB. In some embodiments, the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap includes the first ten amino acids found at the N-terminus of extracellular ActRIIA joined to the ninth amino acid of extracellular ActRIIB. The extracellular ActRII chimera included in the ActRII chimera ligand trap may also include one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIB compared to wild-type extracellular ActRIIB (e.g., SEQ ID NO: 74 shown above), and one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIA compared to wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73 shown above). Amino acid substitutions at 9 different positions may be introduced into an extracellular ActRII chimera (Table 16). An extracellular ActRII chimera may have one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions relative the sequence of a wild-type sequence (e.g., relative to the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 74) if the portion of the chimera corresponds to a region of wild-type extracellular ActRIIB, or relative to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73) if the portion of the chimera corresponds to a region of wild-type extracellular ActRIIA). The positions at which amino acid substitutions may be made, as well as the amino acids that may be substituted at these positions, are listed in Table 16. ActRII chimera ligand traps that may be used in the methods described herein include those described in U.S. Patent Application Publication No. US20230079602, the disclosure of which is incorporated herein by reference.

[0249] Amino acid substitutions can alter the activity and / or binding affinity of the extracellular ActRII chimeras. In some embodiments, the extracellular ActRII chimeras bind to activin A, activin B, myostatin, and / or GDF11 with sufficient affinity to compete with endogenous activin receptors for binding to one or more of these ligands. In some embodiments, the extracellular ActRII chimeras have reduced, weak, or no substantial binding to BMP9 (e.g., compared to wild-type ActRIIB). BMP9 binding may be reduced in extracellular ActRII chimeras containing the amino acid sequence TEEN (SEQ ID NO: 76) or TKEN (SEQ ID NO: 77) at positions X5, Xe, X7, and Xs. In some embodiments, BMP9 binding is reduced in extracellular ActRII chimeras containing the amino acid sequence KKDS (SEQ ID NO: 1027) or TKDS (SEQ ID NO: 1028) at positions X5, Xe, X7, and Xa. In some embodiments, a polypeptide including an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 751-793 (e.g., SEQ ID NOs: 772-793)) with the sequence TEEN (SEQ ID NO: 76) at positions X5, Xe, X7, and Xa can have a substitution of the amino acid K for the amino acid E at position Xe. In some embodiments, a polypeptide including an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 751-793 (e.g., SEQ ID NOs: 772-793)) with the sequence TKEN (SEQ ID NO: 77) at positions X5, Xe, X7, and Xa can have a substitution of the amino acid E for the amino acid K at position Xe. The sequences TEEN (SEQ ID NO: 76) and TKEN (SEQ ID NO: 77) can be used interchangeably in the extracellular ActRII chimeras (e.g., the chimeras in Tables 16 and 17, e.g., SEQ ID NOs: 751 -793 (e.g., SEQ ID NOs: 772-793)).

[0250] Table 16. Amino acid substitutions in an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 751 -771

[0251] In some embodiments, in ActRII chimeras of SEQ ID NOs: 751-771 (shown in Table 16), Xi is D, X2 is I, F, or E, X3 is N or T, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xa is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 174-216, Xi is D, X2 is I or F, X3 is N, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xa is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 1-21 Xi is D, X2 is F, X3 is N, X4 is E, X5 is K, Xe is K, X7 is D, Xa is S, and X9 is Q.

[0252] In some embodiments, ActRII chimera ligand trap contains the sequence of any one of SEQ ID NOs: 772-793 (Table 17).

[0253] Table 17. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 772-793

[0254] In some embodiments, a polypeptide containing an ActRII chimera of Table 16 or 17 may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 30 pM). In some embodiments, the polypeptide containing an ActRII chimera of Table

[0255] 16 or 17 may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM) The polypeptide containing an ActRII chimera of Table 16 or 17 may also bind to growth and differentiation factor 11 (GDF-11) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher).

[0256] In some embodiments, the ActRII chimera included in the ActRII chimera ligand trap results from the substitution of one or more amino acid sequence corresponding to a p-sheet and, optionally, one or more intervening sequence (e.g., a sequence between the p-sheets), from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA). For example, an ActRII chimera may be produced by replacing one or more amino acid sequence corresponding to a p-sheet and, optionally, one or more or an intervening sequence, in ActRIIB with an amino acid sequence corresponding to the p-sheet and, optionally, the intervening sequence, from ActRIIA. An ActRII chimera may also be produced by replacing one or more amino acid sequence corresponding to a p-sheet and, optionally, one or more intervening sequence, in ActRIIA with an amino acid sequence corresponding to the p-sheet and, optionally, the intervening sequence, from ActRIIB. In the ActRII chimeras, a p-sheet and, optionally, an intervening sequence from one protein is replaced with the corresponding p-sheet and, optionally, the corresponding intervening sequence from the other protein (e.g., the 5thp-sheet from ActRIIA (p5A) can be replaced with the 5thp-sheet from ActRIIB (PSB)). Each ActRII protein has seven p- sheets (pi-p?) and eight intervening sequences (Xi-Xa). The ActRII chimeras include at least one of pia, p2a, p3a, p4a, p5a, or p7a and at least one of pib, p2b, p3b, p4b, p5b, or p7b. Accordingly, an ActRII chimera included in the ActRII chimera ligand trap may have one to five p-sheet substitutions (e.g., 1 , 2, 3, 4, or 5 of pi, p2, p3, p4, p5, and p7 from one ActRII protein may be substituted with the corresponding p-sheet sequence from the other ActRII protein). The ActRII chimera may also have one to seven intervening sequence substitutions (e.g., 1 , 2, 3, 4, 5, 6, or 7 of Xi, X2, X3, X5, Xe, X7, and Xs from one ActRII protein may be substituted with the corresponding intervening sequence from the other ActRII protein). In some embodiments, the p-sheet sequence that is substituted is a minimal p-sheet sequence (e.g., at least HCFATWK (SEQ ID NO: 805), which is a portion of RHCFATWKNI (p3a) (SEQ ID NO: 804); at least HCYASWR (SEQ ID NO: 807), which is a portion of LHCYASWRNS (p3b) (SEQ ID NO: 806); at least EIVKQGCW (SEQ ID NO: 809), which is a portion of SIEIVKQGCW (p4a) (SEQ ID NO: 808); at least ELVKKGCW (SEQ ID NO: 811), which is a portion of TIELVKKGCW (p4b) (SEQ ID NO: 810); at least VE, which is a portion of VEK (p5a); at least V, which is a portion of VAT (p5b); at least SYF, which is a portion of KFSYF (p7a) (SEQ ID NO: 819); or at least T, which is a portion of RFTHL (p7b) (SEQ ID NO: 820)). The extracellular ActRII chimeras are the same length (e.g., have the same number of amino acids) as wild-type extracellular ActRIIA and ActRIIB, therefore, in embodiments in which minimal p-sheet sequences are substituted, contiguous amino acids from ActRIIA or ActRIIB are used to connect the minimal p-sheet to the neighboring intervening sequences to maintain the length (e.g., the number of amino acids) of the ActRII chimeras (e.g., to prevent the extracellular ActRII chimeras from having fewer amino acids than the corresponding regions of extracellular ActRIIA and ActRIIB). Exemplary ActRII chimera sequences that can be included in an ActRII chimera ligand trap are provided in Table 18. ActRII chimera ligand traps that may be used in the methods described herein include those described in International Patent Application Publication Nos. WQ2022235620 and WQ2024102906, the disclosures of which are incorporated herein by reference. Table 18. Extracellular ActRII chimera sequences

[0257] In some embodiments, the ActRII chimera has the sequence of an ActRII chimera listed in Table 9, below. Table 19. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 1029-1059

[0258] In some embodiments, a polypeptide containing an extracellular ActRII chimera of Table 19 may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 300 pM and about 1 pM). In some embodiments, the polypeptide containing an extracellular ActRII chimera of Table 19 may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 200 pM and about 1 pM, or a KD of less than 1 pM). The polypeptide containing an extracellular ActRII chimera of Table 19 may also bind to growth and differentiation factor 11 (GDF-11) with a KD of approximately 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 200 pM and about 1 pM, or a KD of less than 1 pM). The polypeptide containing an extracellular ActRII chimera of Table 19 may bind to GDF-8 with a KD of approximately 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM). In some embodiments, the polypeptide containing an extracellular ActRII chimera of Table 19 may bind to human BMP9 with a KD of about 1 pM or higher (e.g., a KD of about 1 , 5, 15, 30, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pM or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nM or higher, e.g., a KD of 1 nM or higher). The polypeptide containing an extracellular ActRII chimera of Table 19 may also bind to human BMP10 with a KD of about 1 pM or higher (e.g., a KD of about 1 , 5, 15, 30, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pM or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nM or higher).

[0259] In some embodiments, the extracellular ActRII chimeras described herein have an N-terminal truncation of 1-9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). The N-terminal truncation can involve the removal of 1-9 amino acids from the N-terminus of any of the chimeras shown in Tables 16- 19. The N-terminal truncation can remove amino acids up two to amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimera ligand traps).

[0260] The extracellular ActRII chimera ligand traps may further include a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension can add one or more additional amino acids at the C-terminus (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) to any of the chimeras shown in Tables 16-19. The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA or ActRIIB. For example, C-terminal extensions that can be included in the extracellular ActRII chimera ligand traps of the invention are the amino acid sequence NP and the amino acid sequence NPVTPK (SEQ ID NO: 78), which correspond to sequence found in the same position in wildtype ActRIIA. In some embodiments, an extracellular ActRII chimera ligand trap may further include a moiety (e.g., Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin), which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRII chimera by way of a linker or other covalent bonds. An ActRII chimera ligand trap including an extracellular ActRII chimera fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.

[0261] In some embodiments, a polypeptide containing an ActRII chimera described herein (e.g., an ActRII chimera-Fc fusion protein) has a serum half-life of at least 7 days in humans. The polypeptide containing an ActRII chimera described herein may bind to activin A with a KD of 1 pM or higher (e.g., 10 pM or higher). In some embodiments, the polypeptide containing an ActRII chimera described herein binds to activin A, activin B, and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., compared to wild-type extracellular ActRIIB). In some embodiments, the polypeptide containing an ActRII chimera described herein does not substantially bind to human BMP9.

[0262] Fc domains

[0263] In some embodiments, an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer of an immunoglobulin or a fragment of an Fc domain to increase the serum half-life of the polypeptide. An ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which form an Fc domain in the dimer. As conventionally known in the art, an Fc domain is the protein structure that is found at the C-terminus of an immunoglobulin. An Fc domain includes two Fc domain monomers that are dimerized by the interaction between the CH3 antibody constant domains. An Fc domain forms the minimum structure that binds to an Fc receptor, e.g., FcyRI, FcyRlla, FcyRllb, FcyRllla, FcyRlllb, FcyRIV. In some embodiments, an Fc domain may be mutated to lack effector functions, typical of a “dead” Fc domain. For example, an Fc domain may include specific amino acid substitutions that are known to minimize the interaction between the Fc domain and an Fey receptor. In some embodiments, an Fc domain is from an IgG 1 antibody and includes amino acid substitutions L234A, L235A, and G237A. In some embodiments, an Fc domain is from an lgG1 antibody and includes amino acid substitutions D265A, K322A, and N434A. The aforementioned amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The Kabat numbering of amino acid residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Furthermore, in some embodiments, an Fc domain does not induce any immune system- related response. For example, the Fc domain in a dimer of an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and an Fey receptor. The sequence of an Fc domain monomer that may be fused to an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is shown below (SEQ ID NO: 97): THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0264] The sequence of a wild-type Fc domain monomer that may be fused to an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is shown below in SEQ ID NO: 84:

[0265] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0266] In some embodiments, the Fc domain monomer fused to an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof lacks a terminal lysine. An exemplary sequence for a wild-type Fc domain monomer lacking the terminal lysine is provided below (SEQ ID NO: 79):

[0267] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0268] In some embodiments, an Fc domain monomer is from an IgG 1 antibody and includes amino acid substitutions L12A, L13A, and G15A, relative to the sequence of SEQ ID NO: 97. In some embodiments, an Fc domain monomer is from an IgG 1 antibody and includes amino acid substitutions D43A, K100A, and N212A, relative to the sequence of SEQ ID NO: 97. In some embodiments, the terminal lysine is absent from the Fc domain monomer having the sequence of SEQ ID NO: 97 or SEQ ID NO: 84. In some embodiments, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97, SEQ ID NO: 84, or SEQ ID NO: 79) through conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof and the Fc domain monomer. The Fc domain monomer can be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof. The Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain monomer can be an IgG subtype (e.g., lgG1 , lgG2a, lgG2b, lgG3, or lgG4). In some embodiments, the Fc domain monomer is an IgG 1 Fc domain monomer (e.g., a human IgG 1 Fc domain monomer).

[0269] In some embodiments, an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain contains a hinge domain. The Fc domain can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4). The Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.

[0270] Methods of engineering Fc domains that have reduced dimerization are known in the art. In some embodiments, one or more amino acids with large sidechains (e.g., tyrosine or tryptophan) may be introduced to the CH3-CH3 dimer interface to hinder dimer formation due to steric clash. In other embodiments, one or more amino acids with small sidechains (e.g., alanine, valine, or threonine) may be introduced to the CH3-CH3 dimer interface to remove favorable interactions. Methods of introducing amino acids with large or small sidechains in the CH3 domain are described in, e.g., Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication No. 2006 / 0074225, U.S. Patent Nos. 8,216,805 and 5,731 ,168, Ridgway et al. (Protein Eng. 9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26-35, 1997), and Merchant et al. (Nat Biotechnol. 16:677-681 , 1998), all of which are incorporated herein by reference in their entireties.

[0271] In yet other embodiments, one or more amino acid residues in the CH3 domain that make up the CH3-CH3 interface between two Fc domains are replaced with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) such that the interaction becomes electrostatically unfavorable depending on the specific charged amino acids introduced. Methods of introducing charged amino acids in the CH3 domain to disfavor or prevent dimer formation are described in, e.g., Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication Nos. 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111 , all of which are incorporated herein by reference in their entireties.

[0272] In some embodiments of the invention, an Fc domain includes one or more of the following amino acid substitutions:T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351 H, L351 N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I, relative to the sequence of human IgG 1 . In some embodiments, the terminal lysine is absent from the Fc domain amino acid sequence. In one particular embodiment, an Fc domain includes the amino acid substitution T366W, relative to the sequence of human IgG 1 .

[0273] Albumin-binding peptides

[0274] In some embodiments, an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to a serum protein-binding peptide. Binding to serum protein peptides can improve the pharmacokinetics of protein pharmaceuticals.

[0275] As one example, albumin-binding peptides that can be used in the methods and compositions described here are generally known in the art. In one embodiment, the albumin binding peptide includes the sequence DICLPRWGCLW (SEQ ID NO: 83).

[0276] In the present invention, albumin-binding peptides may be joined to the N- or C-terminus (e.g., C- terminus) of an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular ActRIIA variant. In some embodiments, an albumin-binding peptide is joined, either directly or through a linker, to the N- or C- terminus of an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof.

[0277] In some embodiments, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- or C-terminus of albumin-binding peptide (e.g., SEQ ID NO: 83) through conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof and the albumin-binding peptide. Without being bound to a theory, it is expected that inclusion of an albumin-binding peptide in an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein may lead to prolonged retention of the therapeutic protein through its binding to serum albumin.

[0278] Fibronectin domains

[0279] In some embodiments, an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to fibronectin domains. Binding to fibronectin domains can improve the pharmacokinetics of protein pharmaceuticals.

[0280] Fibronectin domain is a high molecular weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to, e.g., membrane-spanning receptor proteins such as integrins and extracellular matrix components such as collagens and fibrins. In some embodiments of the present invention, a fibronectin domain is joined to the N- or C-terminus (e.g., C-terminus) of an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof. A fibronectin domain can be joined, either directly or through a linker, to the N- or C-terminus of an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof.

[0281] As one example, fibronectin domains that can be used in the methods and compositions described here are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain (SEQ ID NO: 82, below) having amino acids 610-702 of the sequence of UniProt ID NO: P02751.

[0282] GPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIK GLKPGWYEGQLISIQQYGHQEVTRFDFTTTST

[0283] In another embodiment, the fibronectin domain is an adnectin protein.

[0284] In some embodiments, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- or C-terminus of a fibronectin domain (e.g., SEQ ID NO: 82) through conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof and the fibronectin domain. Without being bound to a theory, it is expected that inclusion of a fibronectin domain in an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein may lead to prolonged retention of the therapeutic protein through its binding to integrins and extracellular matrix components such as collagens and fibrins.

[0285] Serum albumin

[0286] In some embodiments, an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to serum albumin. Binding to serum albumins can improve the pharmacokinetics of protein pharmaceuticals.

[0287] Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and constitutes about half of the blood serum proteins. It is monomeric and soluble in the blood. Some of the most crucial functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining osmotic pressure needed for proper distribution of bodily fluids between blood vessels and body tissues. In preferred embodiments, serum albumin is human serum albumin. In some embodiments of the present invention, a human serum albumin is joined to the N- or C-terminus (e.g., C-terminus) of an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof. A human serum albumin can be joined, either directly or through a linker, to the N- or C-terminus of an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof.

[0288] As one example, serum albumins that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the serum albumin includes the sequence of UniProt ID NO: P02768 (SEQ ID NO: 81 , below).

[0289] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEP ERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVV LNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

[0290] In some embodiments, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- or C-terminus of a human serum albumin (e.g., SEQ ID NO: 81) through conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof and the human serum albumin. Without being bound to a theory, it is expected that inclusion of a human serum albumin in an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein may lead to prolonged retention of the therapeutic protein.

[0291] Linkers

[0292] An ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety by way of a linker. In some embodiments, the moiety increases stability of the polypeptide. Exemplary moieties include an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin. In the present invention, a linker between a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97, SEQ ID NO: 84, or SEQ ID NO: 79), an Fc domain, an albumin-binding peptide (e.g., SEQ ID NO: 83), a fibronectin domain (e.g., SEQ ID NO: 82), or a human serum albumin (e.g., SEQ ID NO: 81)) and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6- 72)), can be an amino acid spacer including 1-200 amino acids. Suitable peptide spacers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 98), GGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 100), GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), or SGGG (SEQ ID NO: 107). In some embodiments, a spacer can contain 2 to 12 amino acids including motifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, a spacer can contain 3 to 12 amino acids including motifs of GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). In yet some embodiments, a spacer can contain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), e.g., GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In some embodiments, a spacer can contain motifs of GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128) and GGGGSGGGGSGGGGS (SEQ ID NO: 129). In some embodiments of the invention, an amino acid spacer between a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin) and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6- 72)) may be GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132). In some embodiments, a spacer can also contain amino acids other than glycine, alanine, and serine, e.g., AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 96), KESGSVSSEQLAQFRSLD (SEQ ID NO: 95), GENLYFQSGG (SEQ ID NO: 94), SACYCELS (SEQ ID NO: 93), RSIAT (SEQ ID NO: 92), RPACKIPNDLKQKVMNH (SEQ ID NO: 91), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 90), AAANSSIDLISVPVDSR (SEQ ID NO: 89), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 88). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO: 87). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of prolinerich sequences such as (XP)n, in which X may be any amino acid (e.g., A, K, or E) and n is from 1-5, and PAPAP (SEQ ID NO: 86).

[0293] The length of the peptide spacer 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 spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0294] In some embodiments, the linker between a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97, SEQ ID NO: 84, or SEQ ID NO: 79), an Fc domain, an albumin-binding peptide (e.g., SEQ ID NO: 83), a fibronectin domain (e.g., SEQ ID NO: 82), or a human serum albumin (e.g., SEQ ID NO: 81)) and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), is an amino acid spacer having the sequence GGG. For example, an ActRIIA ligand trap for use as described herein can contain an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 6-72) fused to an Fc domain monomer (e.g., SEQ ID NO: 79) by a GGG linker. An exemplary polypeptide containing an ActRIIA variant of SEQ ID NO: 69, a GGG linker, and an Fc domain monomer lacking a terminal lysine (SEQ ID NO: 79) is provided below (SEQ ID NO: 80):

[0295] GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKG CWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0296] In another example, a polypeptide for use as described herein can contain an extracellular ActRIIB variant (e.g., any one of SEQ ID NOs: 731-744) fused to an Fc domain monomer (e.g., SEQ ID NO: 84 or SEQ ID NO: 79) by a GGG linker. An exemplary polypeptide containing an ActRIIB variant of SEQ ID NO: 744, a GGG linker, and an Fc domain monomer (SEQ ID NO: 84) is provided below (SEQ ID NO: 830):

[0297] GRGEAETRECLYYNANWELERTNQSGVERCEGEKDKRLHCYASWRNSSGSLEIVKKG CWLDDFNCYDRDTCVATKENPQVYFCCCEGNMCNERFTHLPEAGGPEVTYEPPPTAP TGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0298] In another example, a polypeptide for use as described herein can contain an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 772-793) fused to an Fc domain monomer (e.g., SEQ ID NO: 84 or SEQ ID NO: 79) by a GGG linker. An exemplary polypeptide containing an ActRII chimera of SEQ ID NO: 791 , a GGG linker, and an Fc domain monomer (SEQ ID NO: 84) is provided below (SEQ ID NO: 831):

[0299] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQ GCWLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0300] An exemplary polypeptide containing an ActRII chimera of SEQ ID NO: 775, a GGG linker, and an Fc domain monomer (SEQ ID NO: 84) is provided below (SEQ ID NO: 832):

[0301] GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQGC WLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0302] An exemplary polypeptide containing an ActRII chimera of SEQ ID NO: 790, a GGG linker, and an Fc domain monomer (SEQ ID NO: 84) is provided below (SEQ ID NO: 833):

[0303] GAILGRAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQG CWLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0304] The C-terminal Lys339 of the polypeptide of SEQ ID NO: 831 or SEQ ID NO: 833 (the C-terminal Lys in the Fc region of SEQ ID NO: 831 or SEQ ID NO: 833), the C-terminal Lys337 of the polypeptide of SEQ ID NO: 832 (the C-terminal Lys in the Fc region of SEQ ID NO: 832) and the C-terminal Lys345 of the polypeptide of SEQ ID NO: 830 (the C-terminal Lys in the Fc region of SEQ ID NO: 830) may or may not be present, without affecting the structure or stability of the polypeptide. The disclosure specifically contemplates SEQ ID NOs: 831 and 833 that do not include the C-terminal Lys corresponding to Lys339, SEQ ID NO: 832 that does not include the C-terminal Lys corresponding to Lys337, and SEQ ID NO: 830 that does not include the C-terminal Lys corresponding to Lys345. The polypeptide of SEQ ID NO: 831 or SEQ ID NO: 833 may be expressed including a C-terminal Lys339 which then may be proteolytically cleaved upon expression of the polypeptide, the polypeptide of SEQ ID NO: 832 may be expressed including a C-terminal Lys337 which then may be proteolytically cleaved upon expression of the polypeptide, and the polypeptide of SEQ ID NO: 830 may be expressed including a C-terminal Lys345 which then may be proteolytically cleaved upon expression of the polypeptide (e.g., the polypeptides of SEQ ID NOs: 830-833 are expressed using nucleic acid constructs encoding the polypeptide including a C-terminal lysine residue). The polypeptides of SEQ ID NOs: 831 , 833, 832, and 830 may also be expressed without including the C-terminal Lys339, the C-terminal Lys337, and the C-terminal Lys345, respectively.

[0305] Vectors, host cells, and protein production

[0306] The ActRII signaling inhibitors of the invention can be produced from a host cell. A host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like). The choice of nucleic acid vectors depends in part on the host cells to be used. Generally, preferred host cells are of either eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0307] Nucleic acid vector construction and host cells

[0308] A nucleic acid sequence encoding the amino acid sequence of a polypeptide of the invention (i.e., an ActRII signaling inhibitor) may be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. A nucleic acid molecule encoding a polypeptide of the invention may be obtained using standard techniques, e.g., gene synthesis. Alternatively, for the production of ActRII ligand traps, a nucleic acid molecule encoding a wild-type portion of extracellular ActRIIA or ActRIIB may be mutated to include specific amino acid substitutions using standard techniques in the art, e.g., QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0309] A nucleic acid sequence encoding a polypeptide of the invention may be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purpose of the invention. Each vector may include various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding protein of interest, and a transcription termination sequence.

[0310] In some embodiments, mammalian cells may be used as host cells for the invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells may also be used as host cells for the invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC®31 ,446), E. coli A 1776 (ATCC®31 ,537, E. coli BL21 (DE3) (ATCC® BAA- 1025), and E. coli RV308 (ATCC® 31 ,608). Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems may be chosen to ensure the correct modification and processing of the polypeptide expressed. The above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein production, host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Methods for expression of therapeutic proteins are known in the art, see, for example, Paulina Baibas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012.

[0311] Protein production, recovery, and purification

[0312] Host cells used to produce the polypeptides of the invention may be grown in media known in the art and suitable for culturing of the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10%. The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.

[0313] In some embodiments, depending on the expression vector and the host cells used, the expressed protein may be secreted from the host cells (e.g., mammalian host cells) into the cell culture media. Protein recovery may involve filtering the cell culture media to remove cell debris. The proteins may be further purified. A polypeptide of the invention may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, the protein can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column (e.g., POROS Protein A chromatography) with chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting-out and dialysis procedures.

[0314] In other embodiments, host cells may be disrupted, e.g., by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, cell debris may be removed by centrifugation or filtration. In some instances, a polypeptide can be conjugated to marker sequences, such as a peptide to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His- tag), which binds to nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0315] Alternatively, the polypeptides of the invention can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administrating a vector (such as a viral vector (e.g., a retroviral vector, adenoviral vector, poxviral vector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, and alphaviral vector)) containing a nucleic acid molecule encoding the polypeptide of the invention. The vector, once inside a cell of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.) will promote expression of the polypeptide, which is then secreted from the cell. If treatment of a disease or disorder is the desired outcome, no further action may be required. If collection of the protein is desired, blood may be collected from the subject and the protein purified from the blood by methods known in the art.

[0316] Pharmaceutical compositions and preparations

[0317] The invention features pharmaceutical compositions that include the polypeptides described herein (e.g., an ActRII signaling inhibitor, such as an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, a pharmaceutical composition of the invention includes an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1- 70 (e.g., SEQ ID NOs: 6-70)) with a C-terminal extension (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) as the therapeutic protein. In some embodiments, a pharmaceutical composition of the invention includes an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1- 72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety (e.g., Fc domain monomer, or a dimer thereof, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin) as the therapeutic protein. In some embodiments, a pharmaceutical composition of the invention including a polypeptide of the invention may be used in combination with other agents (e.g., therapeutic biologies and / or small molecules) or compositions in a therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art. In some embodiments, a pharmaceutical composition of the invention includes a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding a polypeptide of the invention, or a vector containing such a nucleic acid molecule.

[0318] Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, arginine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol. Pharmaceutical compositions of the invention can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a-MEM), F-12 medium). Formulation methods are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor & Francis Group, CRC Press (2015).

[0319] The pharmaceutical compositions of the invention may be prepared in microcapsules, such as hydroxylmethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule. The pharmaceutical compositions of the invention may also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and Practice of Pharmacy 22ndedition (2012). The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0320] The pharmaceutical compositions of the invention may also be prepared as a sustained-release formulation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides of the invention. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and y ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™, and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations enable release of molecules over a few months, e.g., one to six months, while other formulations release pharmaceutical compositions of the invention for shorter time periods, e.g., days to weeks.

[0321] The pharmaceutical composition may be formed in a unit dose form as needed. The amount of active component, e.g., a polypeptide of the invention, included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01- 100 mg / kg of body weight).

[0322] The pharmaceutical composition for gene therapy can be in an acceptable diluent or can include a slow-release matrix in which the gene delivery vehicle is imbedded. If hydrodynamic injection is used as the delivery method, the pharmaceutical composition containing a nucleic acid molecule encoding a polypeptide described herein or a vector (e.g., a viral vector) containing the nucleic acid molecule is delivered rapidly in a large fluid volume intravenously. Vectors that may be used as in vivo gene delivery vehicle include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara), adeno-associated viral vectors, and alphaviral vectors.

[0323] Routes, dosage, and administration

[0324] Pharmaceutical compositions that include the polypeptides of the invention as the therapeutic proteins may be formulated for, e.g., intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical composition may also be formulated for, or administered via, oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, e.g., ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).

[0325] In some embodiments, a pharmaceutical composition that includes a nucleic acid molecule encoding a polypeptide of the invention or a vector containing such nucleic acid molecule may be administered by way of gene delivery. Methods of gene delivery are well-known to one of skill in the art. Vectors that may be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vectors, and alphaviral vectors. In some embodiments, mRNA molecules encoding polypeptides of the invention may be administered directly to a subject.

[0326] In some embodiments of the present invention, nucleic acid molecules encoding a polypeptide described herein or vectors containing such nucleic acid molecules may be administered using a hydrodynamic injection platform. In the hydrodynamic injection method, a nucleic acid molecule encoding a polypeptide described herein is put under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). The plasmid is often delivered rapidly in a large fluid volume intravenously. Hydrodynamic injection uses controlled hydrodynamic pressure in veins to enhance cell permeability such that the elevated pressure from the rapid injection of the large fluid volume results in fluid and plasmid extravasation from the vein. The expression of the nucleic acid molecule is driven primarily by the liver. In mice, hydrodynamic injection is often performed by injection of the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding a polypeptide described herein may be administered using hydrodynamic injection.

[0327] The dosage of the pharmaceutical compositions of the invention depends on factors including the route of administration, the disease to be treated, and physical characteristics, e.g., age, weight, general health, of the subject. A pharmaceutical composition of the invention may include a dosage of an ActRII signaling inhibitor of the invention ranging from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg) and, in a more specific embodiment, about 0.1 to about 30 mg / kg and, in a more specific embodiment, about 0.3 to about 30 mg / kg. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.

[0328] The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Generally, therapeutic proteins are dosed at 0.1-100 mg / kg, e.g., 0.5-50 mg / kg. Pharmaceutical compositions that include a polypeptide of the invention may be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 times or more) daily, weekly, biweekly, every four weeks, monthly, bimonthly, quarterly, biannually, annually, or as medically necessary. In some embodiments, pharmaceutical compositions that include a polypeptide of the invention may be administered to a subject in need thereof weekly, biweekly, every four weeks, monthly, bimonthly, or quarterly. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may decrease as the medical condition improves or increase as the health of the patient declines.

[0329] Methods of treatment

[0330] The ActRII signaling inhibitors described herein, such as an ActRII ligand trap (e.g., an ActRIIA ligand trap, an ActRIIB ligand trap, or an ActRII chimera ligand trap, such as an ActRIIA ligand trap containing an extracellular ActRIIA variant described herein), can be used to disrupt endogenous activin signaling. Therefore, the ActRII signaling inhibitors can be used to treat diseases or conditions in which activin signaling has been implicated. For example, activin receptor ligand, GDF11 , has been found to be overexpressed in a mouse model of hemolytic anemia and associated with defects in red blood cell production. Signaling pathways involving activins also regulate hematopoiesis by preventing the differentiation of red blood cell, platelet, and neutrophil progenitor cells in order to maintain progenitor cells in a quiescent state. Without wishing to be bound by theory, a therapeutic agent that binds to activin receptor ligands (e.g., myostatin, activins, and / or GDF11) and reduces their binding to or interaction with endogenous activin receptors (e.g., by sequestering the endogenous ligands) or that binds to the endogenous receptors and disrupts their interactions with these ligands may have therapeutic utility for treating diseases or conditions involving ineffective hematopoiesis, such as cytopenias (e.g., anemia, thrombocytopenia, and / or neutropenia) associated with myelofibrosis or with a cytopenia-associated myelofibrosis treatment. Based on the discovery that administration of a polypeptide containing an ActRIIA variant described herein decreased transfusion burden, increased markers of erythropoiesis, increased hemoglobin, maintained or improved platelet count, reduced spleen size, and reduced disease symptoms in human subjects having anemia and myelofibrosis, the ActRII signaling inhibitors described herein (e.g., an ActRIIA ligand trap including an ActRIIA variant) can be used to treat myelofibrosis, a cytopenia associated with myelofibrosis, or a subject receiving treatment with a cytopenia-associated myelofibrosis treatment in subjects with more severe disease, such as transfusion-dependent subjects.

[0331] The ActRII signaling inhibitors described herein (e.g., an activin A antibody, a myostatin antibody, an activin B antibody, a GDF-11 antibody, an ActRII antibody, or an ActRII ligand trap, such as an ActRIIA ligand trap, an ActRIIB ligand trap, or an ActRII chimera ligand trap, e.g., an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), e.g., an effective amount of an ActRIIA ligand trap including an extracellular ActRIIA variant) can be used to treat a transfusion-dependent (TD) subject having or at risk of developing a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis or to treat myelofibrosis (e.g., to treat multiple facets of the pathology of myelofibrosis) in a TD subject. In some embodiments, the subject is ineligible for treatment with a JAK inhibitor (e.g., ruxolitinib, fedratinib, or pacritinib, for example, due to already having a cytopenia or due to myelofibrosis risk status). In some embodiments, the subject has discontinued treatment with a JAK inhibitor to due relapsed disease following treatment with a JAK inhibitor, being refractory to treatment with a JAK inhibitor, or intolerance to treatment with a JAK inhibitor, or no longer meeting the risk / benefit ratio to continue treatment with the JAK inhibitor. In some embodiments, the subject has a defect in JAK / STAT signaling (e.g., a reduction, deficiency, or failure in JAK / STAT signaling). The ActRII signaling inhibitors described herein (e.g., an activin A antibody, a myostatin antibody, an activin B antibody, a GDF-11 antibody, an ActRII antibody, or an ActRII ligand trap, such as an ActRIIA ligand trap, an ActRIIB ligand trap, or an ActRII chimera ligand trap, e.g., an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), e.g., an effective amount of an ActRIIA ligand trap including an extracellular ActRIIA variant) can also be used to treat a TD subject receiving treatment with a cytopenia-associated myelofibrosis treatment (i.e., the ActRII signaling inhibitor can be administered in combination with the cytopenia-associated myelofibrosis treatment). In some embodiments, the subject has a cytopenia (e.g., anemia, thrombocytopenia, and / or neutropenia) (e.g., the subject has already developed a cytopenia or is identified as having a cytopenia prior to treatment with an ActRII signaling inhibitor described herein, e.g., a cytopenia caused by the cytopenia-associated myelofibrosis treatment). In some embodiments, the subject has not yet developed a cytopenia or is not identified as having a cytopenia when treatment with the ActRII signaling inhibitor is initiated. In some embodiments, the subject is receiving treatment with a cytopenia-associated myelofibrosis treatment for myelofibrosis, such as PMF, post-ET MF, or post-PV MF (e.g., diagnosed according to the 2017 World Health Organization criteria). In some embodiments, the subject is receiving treatment with a cytopenia- associated myelofibrosis treatment for polycythemia vera. In some embodiments, the subject is receiving treatment with a cytopenia-associated myelofibrosis treatment for steroid-refractory acute graft-versus- host disease. In some embodiments, the subject is receiving treatment with a cytopenia-associated myelofibrosis treatment for chronic graft-versus-host disease (e.g., after failure of one or two lines of systemic therapy in an adult subject or a pediatric subject 12 years of age or older). In some embodiments, the subject has been receiving treatment with the cytopenia-associated myelofibrosis treatment for at least eight weeks (e.g., 8 weeks or longer, such as 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18,

[0332] 19, 20, 21 , 22, 23, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months) and has been on a stable dose of the cytopenia-associated myelofibrosis treatment for at least 4 weeks (e.g., 4 weeks or longer, such as 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,

[0333] 20, 21 , 22, 23, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months) prior to co-administration of an ActRII signaling inhibitor described herein. In some embodiments, the subject has been receiving treatment with the cytopenia-associated myelofibrosis treatment for at least eight weeks and less than six months (e.g., 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 weeks) and has been on a stable dose of the cytopenia-associated myelofibrosis treatment for at least 4 weeks (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 weeks) prior to co-administration of an ActRII signaling inhibitor described herein. In some embodiments, the subject has been receiving treatment with the cytopenia-associated myelofibrosis treatment for less than eight weeks (e.g., 7, 6, 5, 4, 3, 2, 1 weeks or shorter) prior to co-administration of an ActRII signaling inhibitor described herein. In some embodiments, treatment with the cytopenia- associated myelofibrosis treatment and the ActRII signaling inhibitor is started concurrently (e.g., the subject begins treatment with both agents at approximately the same time, e.g., begins treatment with both agents during the same day, week, or month).

[0334] In some embodiments, the myelofibrosis is PMF, post-ET MF, or post-PV MF (e.g., diagnosed according to the 2017 World Health Organization criteria). In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance score of less than or equal to two. In some embodiments, the subject has anemia. Anemia is defined as hemoglobin <10 g / dL during screening, or receiving RBC transfusions. In some embodiments, the subject has thrombocytopenia. In some embodiments, the subject has both anemia and thrombocytopenia. In some embodiments, the subject has neutropenia. In some embodiments, the subject has anemia and neutropenia. In some embodiments, the subject has thrombocytopenia and neutropenia. In some embodiments, the subject has anemia, thrombocytopenia, and neutropenia. In some embodiments, the myelofibrosis is intermediate (e.g., intermediate-1 or intermediate-2) or high-risk myelofibrosis, e.g., according to the dynamic international prognostic scoring system (DIPSS). In some embodiments, the subject is identified as having a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) prior to administration of an ActRII signaling inhibitor described herein. In some embodiments, the method includes a step of identifying the subject as having a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) (e.g., by evaluating red blood cell, hemoglobin, hematocrit, platelet, and / or neutrophil levels) prior to administration of an ActRII signaling inhibitor described herein. The method can further include evaluating red blood cell, hemoglobin, hematocrit, reticulocyte, platelet, and / or neutrophil levels after administration of an ActRII signaling inhibitor described herein (e.g., 12 hours, 24 hours, 1 , 2, 3, 4, 5, 6, or 7 days, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with an ActRII signaling inhibitor described herein, such as by taking a CBC). In some embodiments, the subject does not receive concurrent treatment with an erythropoiesis stimulating agent (ESA), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), a thrombopoietin agonist (TPO), an immunomodulator imide drug (I MiD; e.g., thalidomide, pomalidomide, lenalidomide), interferon, or hydroxyurea, danazol, a steroid (other than prednisone of less than or equal to 10 mg / day or corticosteroid equivalent), a cytotoxic or chemotherapeutic agent, a hypomethylating agent, an RBC hematopoietic growth factor (e.g., Interleukin-3), an androgen, an oral retinoid, or arsenic trioxide.

[0335] The ActRII signaling inhibitors described herein (e.g., an activin A antibody, a myostatin antibody, an activin B antibody, a GDF-11 antibody, an ActRII antibody, or an ActRII ligand trap, such as an ActRIIA ligand trap, an ActRIIB ligand trap, or an ActRII chimera ligand trap, e.g., an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), e.g., an effective amount of an ActRIIA ligand trap including an extracellular ActRIIA variant) can also be used to treat splenomegaly associated with extramedullary hematopoiesis (e.g., to reduce spleen volume in a subject having splenomegaly) in a TD subject.

[0336] In some embodiments, the methods described herein affect myostatin, activin A, activin B, and / or BMP9 signaling (e.g., reduce or inhibit the binding of activin A, activin B, myostatin, and / or BMP9 to their endogenous receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII) in the subject. In some embodiments, the methods described herein increase hemoglobin levels, increase hematocrit, increase red blood cell count, increase red blood cell volume, increase red cell mass, increase reticulocytes, increase proerythroblasts, increase or induce red blood cell formation or production, increase the maturation and / or differentiation of erythroid progenitors (early or late- (e.g., terminal) stage progenitors, e.g., early-stage erythroid progenitors, such as burst forming unit-erythroid cells (BFU-Es) and / or colony forming unit-erythroid cells (CFU-Es), e.g., increase the maturation and / or differentiation of BFU-Es and / or CFU-Es into proerythroblasts, reticulocytes, or red blood cells, e.g., increase proerythroblast and / or reticulocyte numbers), increase late-stage erythroid precursor maturation (e.g., terminal maturation, such as the maturation of reticulocytes into red blood cells, or the maturation of erythroblasts into reticulocytes and / or red blood cells), recruit early-stage progenitors into the erythroid lineage, increase the number of early- stage erythroid precursors and / or progenitors (e.g., expand the early-stage precursor population to provide a continuous supply of precursors to replenish polychromatic erythroblasts and allow for a continuous supply of maturing reticulocytes), promote the progression of erythroid precursors and / or progenitors through erythropoiesis, reduce the accumulation of red blood cell progenitor cells (e.g., by stimulating progenitor cells to progress to maturation), increase platelet levels (e.g., increase platelet count), increase or induce megakaryocyte differentiation and / or maturation (e.g., to produce platelets, e.g., terminal maturation of pro-platelets to platelets), reduce platelet progenitor accumulation (e.g., by stimulating progenitor cells to progress to maturation), increase megakaryocyte progenitors (e.g., increase megakaryocyte progenitor renewal), increase pro-platelets, promote or increase platelet formation or production, increase neutrophil levels (e.g., increase neutrophil count), increase or induce the differentiation and / or maturation of progenitor cells (e.g., myeloid progenitors, myeloblasts, or myelocytes) into neutrophils, and / or induce or increase neutrophil formation or production in the subject. In some embodiments, the methods described herein increase the rate of recovery from thrombocytopenia. These changes may be observed in a subject treated with an ActRII signaling inhibitor described herein (e.g., an activin A antibody, a myostatin antibody, an activin B antibody, a GDF-11 antibody, an ActRII antibody, or an ActRII ligand trap, such as an ActRIIA ligand trap, an ActRIIB ligand trap, or an ActRII chimera ligand trap, e.g., an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), e.g., an effective amount of an ActRIIA ligand trap including an extracellular ActRIIA variant) compared to measurements obtained prior to treatment or compared to measurements obtained from untreated subjects having the same disease or condition (e.g., myelofibrosis or a myelofibrosis- associated cytopenia). In some embodiments, the methods described herein reduce spleen size (e.g., in a subject having an enlarged spleen, such as an enlarged spleen associated with myelofibrosis). In some embodiments, the methods described herein improve or restore hematopoiesis in the bone marrow, reduce or reverse reticulin and / or collagen deposition, or reverse bony changes associated with myelofibrosis. In some embodiments, the methods described herein reduce or ameliorate megakaryocyte dysfunction (e.g., megakaryocyte dysfunction in the bone marrow in a subject with myelofibrosis), which may prevent or reduce inflammation / fibrosis, restore hematopoiesis in the bone marrow, and treat cytopenias due to myelofibrosis and those that result from JAK inhibitor treatment. In some embodiments, the methods described herein reduce or resolve hepatosplenomegaly or splenomegaly (e.g., reduce spleen volume and / or splenic extra medullary hematopoiesis) and its symptoms in a subject with myelofibrosis. In some embodiments, the methods described herein reduce bone marrow fibrosis and alleviate the symptoms caused by the loss of bone marrow function. In some embodiments, the methods described herein slow or reduce the progression of bone marrow fibrosis. In some embodiments, the methods described herein improve or ameliorate the attenuated bone resorption and osteosclerosis in patients with myelofibrosis. In some embodiments, the methods described herein improve fibrosis, bone histomorphology, spleen size (e.g., reduce spleen size), myelofibrosis symptoms, bone marrow fibrosis, and / or osteosclerotic dysplasia in a subject with myelofibrosis. In some embodiments, the methods described herein increase body weight. In some embodiments, the methods described treat or reduce cachexia. In some embodiments, the methods described treat or reverse a cytopenia (e.g., anemia, thrombocytopenia, and / or neutropenia) caused by a cytopenia-associated myelofibrosis treatment and reverse reductions in red blood cells, platelets, and / or neutrophils induced by the cytopenia-associated myelofibrosis treatment. In some embodiments, the methods described herein improve constitutional symptoms associated with myelofibrosis. In some embodiments, the methods described herein reduce bleeding events. In some embodiments, the methods described herein decrease infections.

[0337] In some embodiments, treatment according to methods described herein leads to a mean hemoglobin increase of greater than or equal to 1 .5 g / dL or 2.0 g / dL from baseline or pretreatment measurements over a period of 12 consecutive weeks or more, such as 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24, weeks, 26 weeks, 1 year, 2 years or more, during treatment with an ActRII signaling inhibitor described herein, for example during the first 24 weeks or 52 weeks of treatment of a transfusion-dependent (TD) subject according to the methods described herein. In some embodiments, treatment according to methods described herein leads to a decrease of one or more in the brief fatigue inventory score from baseline within the first 24 weeks or 52 weeks of treatment of a TD subject according to the methods described herein. In some embodiments, the methods described herein reduce the need of a subject, such as a subject with anemia and myelofibrosis requiring RBC transfusions, for a blood transfusion (e.g., reduce transfusion burden, for example, the subject no longer needs blood transfusions, or the subject needs less frequent blood transfusion than before treatment with the compositions and methods described herein). In some embodiments, treatment according to the methods described herein reduces the number of RBC transfusions from baseline pre-treatment measurements (e.g., measurements taken over 12 weeks directly preceding treatment initiation with an ActRII signaling inhibitor described herein) for a period of 12 consecutive weeks of more, such as 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24, weeks, 26 weeks, 1 year, 2 years or more, during treatment with an ActRII signaling inhibitor described herein, for example during the first 24 weeks or 52 weeks of treatment according to the methods described herein. In some embodiments, the compositions and methods described herein promote transfusion independence (e.g., a subject who required 1 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) RBC units over 12 weeks directly preceding treatment initiation with an ActRII signaling inhibitor described herein does not require a transfusion for 12 consecutive weeks or more, such as 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24, weeks, 26 weeks, 1 year, 2 years or more, during treatment with an ActRII signaling inhibitor described herein, for example during the first 24 weeks or 52 weeks of treatment according to the methods described herein). Concurrent treatment for anemia with RBC transfusions is recommended for a subject with myelofibrosis when hemoglobin is < 8.0 g / dL, and may be recommended if Hgb is > 8.0 g / dL and associated with symptom(s) of anemia (e.g., hemodynamic or pulmonary compromise requiring treatment) or comorbidity justifying a threshold > 8.0 g / dL Hgb. A complete blood count (CBC) can be taken to assess the response of a subject to treatment with a composition described herein, and hemoglobin levels can be reviewed to determine whether the subject has a stable hemoglobin level above the transfusion threshold. In subjects who achieve transfusion independence, both hemoglobin levels and absolute reticulocyte counts may increase. In some embodiments, treatment according to the methods described herein leads to an improvement in the Myelofibrosis Symptom Assessment Form Total Symptom Score (MF-SAF-TSS) of greater than or equal to 50% from baseline (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more from baseline), such as by 24 weeks or 52 weeks of treatment according to the methods described herein. In some embodiments, treatment according to the methods described herein leads to a decrease in spleen volume of greater than or equal to 35% from baseline (e.g., at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more from baseline) as measured by computed tomography, such as by 24 weeks or 52 weeks of treatment according to the methods described herein. In some embodiments, the compositions and methods described herein slow or inhibit the progression to acute myeloid leukemia (AML) (bone marrow blasts >20%) and / or accelerated myelofibrosis (bone marrow blasts >10%), such as by 24 weeks or 52 weeks of treatment according to the methods described herein. In some embodiments, treatment according to the methods described herein leads to a mean platelet increase from baseline of greater than 30 x 109 / L for 12 weeks or more, such as 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24, weeks, 26 weeks, 1 year, 2 years or more, during treatment with an ActRII signaling inhibitor described herein (in the absence of platelet transfusions), for example by 24 weeks or 52 weeks of treatment according to the methods described herein. In some embodiments, treatment according to the methods described herein reduces episodes of anemia, neutropenia, and thrombocytopenia of >Grade 1. In some embodiments, treatment according to the methods described herein allows subjects to maintain the dose intensity of the cytopenia-associated myelofibrosis treatment. In some embodiments, treatment according to the methods described herein improves tolerability or adherence to the cytopenia-associated myelofibrosis treatment, for example, the subject can remain on the cytopenia-associated myelofibrosis treatment for 12 weeks, 24 weeks, 52 weeks, or longer with concomitant treatment with an ActRII signaling inhibitor described herein. In some embodiments, treatment according to the methods described herein reduces osteosclerosis from baseline as assessed using CT, such as by 24 weeks or 52 weeks of treatment as described herein. In some embodiments, treatment according to the methods described herein leads to a decrease in Patient Reported Outcomes Measurement Information System (PROMIS) score or BFI score from baseline, such as by 24 weeks or 52 weeks of treatment as described herein. In some embodiments, treatment according to the methods described herein slows or reduces the progression of bone marrow fibrosis or improves (e.g., reverses) bone marrow fibrosis. For example, treatment according to the methods described herein may lead to an improvement in bone marrow fibrosis grade from baseline or may prevent bone marrow fibrosis grade from worsening, such as by 24 weeks or 52 weeks of treatment as described herein. Treatment according to the methods described herein may also increase red cell parameters, such as reticulocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and reticulocyte cell hemoglobin, and / or biomarkers of blood cell production, such as erythropoietin (EPO) and thrombopoietin (TPO) levels. In some embodiments, treatment according to the methods described herein increases mean platelet volume and immature platelet fraction. In some embodiments, treatment according to the methods described herein increases FSH compared to baseline. In some embodiments, treatment according to the methods described herein increases biomarkers of bone metabolism compared to baseline, such as bone specific alkaline phosphatase (BSAP) and serum C-telopeptide of type I collagen (CTX). In some embodiments, treatment according to the methods described herein reduces the development of myelofibrosis-associated molecular and cytogenic abnormalities over the duration of treatment.

[0338] Treatment according to the methods described herein may also lead to changes in biomarkers of iron metabolism (e.g., serum iron, ferritin, transferrin, transferrin saturation, total iron binding capacity, soluble transferrin receptor level, and hepcidin), dose of iron chelators, and cytokine levels compared to baseline, such as by 8 weeks, 12 weeks, 20 weeks, 24 weeks, 26 weeks, 52 weeks, or 2 years of treatment as described herein. For example, treatment according to the methods described herein (e.g., treatment with an ActRII signaling inhibitor described herein, such as an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can lead to a reduction in serum ferritin and in increase in soluble transferrin receptor (sTfR). Ferritin is expected to decrease as patients become transfusionindependent and ineffective erythropoiesis improves, as iron stores are incorporated into new red blood cells. An increase in sTfR is a surrogate marker for induction of erythropoiesis, and measurement of sTfR may be used as a pharmacodynamic marker to monitor changes in erythropoiesis in response to therapy, potentially even before changes in hemoglobin are apparent.

[0339] In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage.

[0340] In some embodiments the ActRII signaling inhibitor that is used in the methods described herein is an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, the ActRIIA ligand trap including an extracellular ActRIIA variant is administered at a dosage ranging from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg) and, in a more specific embodiment, about 0.1 to about 30 mg / kg and, in a more specific embodiment, about 0.3 to about 30 mg / kg. In any of the methods described herein an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs: 6-71)) that further includes a C- terminal extension of one or more amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids) may be used as the therapeutic protein. In any of the methods described herein, a dimer (e.g., homodimer or heterodimer) formed by the interaction of two Fc domain monomers that are each fused to a polypeptide including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be used as the therapeutic protein. In any of the methods described herein, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6- 72)) fused to a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or a human serum albumin) may be used as the therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing said nucleic acids can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector can be administered as part of a pharmaceutical composition.

[0341] Compositions that can be administered to a subject according to the methods described herein are provided in Tables 19-22, below. Combination therapy

[0342] An ActRII signaling inhibitor described herein may be administered to the subject in combination with a cytopenia-associated myelofibrosis treatment. The cytopenia-associated myelofibrosis treatment may be, e.g., ruxolitinib (JAKAFIO / JAKAVI®), fedratinib (INREBIC®), pacritinib (VONJO™), or imetelstat. The cytopenia-associated myelofibrosis treatment may be administered at the same time (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes or less) as the ActRII signaling inhibitor. The agents can also be administered simultaneously via co-formulation. The ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can also be administered sequentially, such that the action of the two overlaps and their combined effect is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each of the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can be performed by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, local routes, and direct absorption through mucous membrane tissues. The ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can be administered by the same route or by different routes. For example, an ActRII signaling inhibitor may be administered by subcutaneous (e.g., for an ActRII ligand trap) or intravenous (e.g., for an Activin A, Activin B, myostatin, GDF-11 , or ActRII antibody) injection or infusion while the cytopenia-associated myelofibrosis treatment can be administered orally (for ruxolitinib, fedratinib, or pacritinib) or by intravenous injection or infusion (for imetelstat). The ActRII signaling inhibitor may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours or up to 1-7, 1-14, 1- 21 or 1-30 days before or after the cytopenia-associated myelofibrosis treatment. In some embodiments, the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment are administered at different frequencies. For example, the ActRII signaling inhibitor can be administered once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months and the cytopenia-associated myelofibrosis treatment can be administered once or twice daily (e.g., for ruxolitinib, fedratinib, or pacritinib). In some embodiments, the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment are administered at the same or at similar frequencies. For example, both the ActRII signaling inhibitor and the cytopenia- associated myelofibrosis treatment can be administered once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months (e.g., when the cytopenia-associated myelofibrosis treatment is imetelstat).

[0343] In some embodiments, the cytopenia-associated myelofibrosis treatment is administered as indicated on the label. For example, ruxolitinib can be administered at a starting dose of 20 mg orally twice daily for subjects with myelofibrosis and greater than 200 x 109 / L platelets at baseline, 15 mg orally twice daily for subjects with myelofibrosis and 100 x 109 / L to 200 x 109 / L platelets at baseline, and 5 mg orally twice daily for subjects with myelofibrosis and 50 x 109 / L to less than 100 x 109 / L platelets at baseline, 10 mg orally twice daily for polycythemia vera, or 5 mg orally twice daily for acute graft-versus- host disease, which can be increased to 10 mg twice daily after at least three days of treatment. In some embodiments, ruxolitinib is administered at a dose of 10 mg / day to 50 mg / day (e.g., 10 mg / day, 15 mg / day, 20 mg / day, 25 mg / day, 30 mg / day, 35 mg / day, 40 mg / day, 45 mg / day, or 50 mg / day). When administered alone, dosing may need to be reduced or discontinued due to the development of cytopenias, but, when administered in combination with an ActRII signaling inhibitor, the subject may be able to remain on the same dose of ruxolitinib with few to no treatment discontinuations and may be able to receive a higher dose of ruxolitinib (e.g., by 5 mg or more, e.g., 5 mg, 10 mg, or 15 mg) compared to the dose of ruxolitinib when it is administered alone. Fedratinib may be taken at a dose of 400 mg or less once daily, such as 400 mg, 300 mg, 200 mg, or 100 mg once daily by subjects with myelofibrosis (e.g., for example, by subjects with intermediate-2 or high-risk primary or secondary myelofibrosis and greater than 50 x 109 / L platelets at baseline). When administered alone, dosing may need to be reduced or discontinued due to the development of cytopenias, but, when administered in combination with an ActRII signaling inhibitor, the subject may be able to remain on the same dose of fedratinib with few to no treatment discontinuations. Pacritinib may be taken at a dose of 200 mg twice daily by subjects with myelofibrosis (e.g., by subjects with intermediate or high-risk primary or secondary (post-polycythemia vera or post-essential thrombocythemia) myelofibrosis with a platelet count below 50 x 109 / L platelets at baseline), which may be reduced to 100 mg twice daily or 100 mg once daily if dose modification is needed for adverse reactions. When administered alone, dosing may need to be reduced or discontinued due to the development of cytopenias, but, when administered in combination with an ActRII signaling inhibitor, the subject may be able to remain on the same dose of pacritinib with few to no treatment discontinuations. Imetelstat may be administered by intravenous infusion at a dose of about 1.0 mg / kg to about 50 mg / kg (e.g., 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0, 9.4, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, or 50.0 mg / kg) once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months. When administered alone, dosing may need to be reduced or discontinued due to the development of cytopenias, but, when administered in combination with an ActRII signaling inhibitor, the subject may be able to remain on the same dose of imetelstat with few to no treatment discontinuations and may be able to receive a higher dose of imetelstat (e.g., by 1 .0 mg / kg or more, e.g., 1 .0 mg / kg, 2.0, mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg or more) compared to the dose of imetelstat when it is administered alone or receive imetelstat treatment less frequently. The ActRII signaling inhibitor can be administered by subcutaneous or intravenous injection or infusion at a dose of from about 0.01 to about 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1 , 1 .25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg) and, in a more specific embodiment, about 0.1 to about 30 mg / kg and, in a more specific embodiment, about 0.3 to about 30 mg / kg, once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months, or once a year.

[0344] In some embodiments, combination therapy with an ActRII signaling inhibitor and a cytopenia- associated myelofibrosis treatment reduces the adverse reactions associated with the cytopenia- associated myelofibrosis treatment, such as the development of anemia, thrombocytopenia, and / or neutropenia that can lead to treatment interruptions and discontinuations. In some embodiments, combination therapy reduces or ameliorates anemia, thrombocytopenia, and / or neutropenia, or reduces the number of episodes of one or more of these cytopenias. In some embodiments, combination therapy improves adherence to treatment with the cytopenia-associated myelofibrosis treatment (e.g., the subject can remain on treatment for a longer period of time), improves tolerability of the cytopenia-associated myelofibrosis treatment (e.g., the subject can remain on the same dose or increase the dose of the cytopenia-associated myelofibrosis treatment), decreases transfusion burden, decreases bleeding events, decreases infections, or decreases interruptions or discontinuations in treatment with the cytopenia-associated myelofibrosis treatment.

[0345] Kits

[0346] An ActRII signaling inhibitor and a cytopenia-associated myelofibrosis treatment described herein can be provided in a kit for use in treating myelofibrosis. Each agent may be provided in unit dosage form, optionally in a pharmaceutically acceptable excipient (e.g., saline), in an amount sufficient to treat myelofibrosis. The kit can further include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the ActRII signaling inhibitor or cytopenia-associated myelofibrosis treatment.

[0347] Table 20

[0348]

[0349] Table 21

[0350] Table 22

[0351] Table 23

[0352] EXAMPLES

[0353] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0354] Example 1 - Effect of ActRIIA / B-hFc in patients with anemia and myelofibrosis

[0355] Patients with primary MF, post-essential thrombocythemia MF, or post-polycythemia vera MF and anemia were enrolled in a multi-center, open-label, two-part, Phase 2 study. Participants in Part 1 received doses of 0.75 mg / kg, 1.5 mg / kg, 3.0 mg / kg, and 4.5 mg / kg of ActRIIA / B-hFc (a homodimer of SEQ ID NO: 80), administered subcutaneously once every 4 weeks in parallel treatment arms: Arm 1A: monotherapy, Arm 1 B: administration of ActRIIA / B-hFc in combination with ruxolitinib. The primary endpoints evaluate safety and tolerability, and secondary and exploratory endpoints evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of ActRIIA / B-hFc with or without ruxolitinib in treating anemia, splenomegaly, and constitutional symptoms. Safety data are presented for the safety population, defined as all participants who received at least one dose of ActRIIA / B-hFc. Changes in markers of hematopoiesis, spleen volume, and symptom scores are presented for participants at Dose Levels 1- 3 (0.75 mg / kg - 3.0 mg / kg); data for Dose Level 4 (4.5 mg / kg) were not included in the analyses due to limited exposure as of the assessment date. For endpoints assessed over a 12-week period (average change in PD markers or reduction in transfusion burden), only participants with 12 weeks of post-baseline data are included. Participants in the study had significant disease burden, with the majority receiving transfusions: 37% were transfusion-dependent (TD) per IWG 2013 criteria (>6 RBC units / 12 wks), and non-TD participants received a median of 3 RBC units / 12wks at baseline. Most participants had splenomegaly and marked splenomegaly was observed even in the combination arm, indicating that ruxolitinib was not providing sufficient control of disease. Demographics and baseline disease characteristics for the safety population are provided in Table 24, below.

[0356] Table 24. Demographics and Baseline Disease Characteristics (Safety Population)

[0357] CALR=calreticulin; DIPSS=dynamic international prognostic scoring system; Hgb=hemoglobin;

[0358] JAK2=Janus kinase 2; MF-SAF-TSS=myelofibrosis-symptom assessment form-total symptom score (version 4.0, 7-item); MPL=myeloproliferative leukemia protein gene; RBC=red blood cell;

[0359] TD=transfusion dependent; U=units; wks=weeks. *Transfusion-dependent is based on IWG 2013 criteria (Teferri et al., Blood 2013) and is defined as receiving 6 RBC units in the 12 weeks prior to first dose with at least one transfusion event in the 4 weeks preceding first dose

[0360] Both TD and non-TD participants had anemia despite high baseline levels of EPO, underscoring the erythropoietic dysfunction in both groups (FIG. 1). Lower levels of sTfR in TD participants reflect greater erythropoietic dysfunction, with elevated EPO unable to drive erythropoiesis (FIG. 1).

[0361] Table 25 shows the exposure to ActRIIA / B-hFc as of the assessment date and Table 26 shows an overview of treatment emergent adverse events (TEAEs). Table 25. ActRIIA / B-hFc Exposure

[0362] *As of the assessment date, all 8 participants (4 in each cohort; 100%) in the Dose Level 4 cohorts (4.5 mg / kg) were ongoing. Table 26. Overview of Treatment Emergent Adverse Events

[0363] DLT=dose-limiting toxicity (see description in text below); SAE=serious adverse event *>10% of all participants (including monotherapy and combination therapy arms)

[0364] ActRIIA / B-hFc was generally well-tolerated in these study participants who had significant disease burden and complex comorbidities at doses ranging from 0.75 to 4.5 mg / kg, both as monotherapy and in combination with ruxolitinib. Treatment-related TEAEs were relatively infrequent, and the majority were Grade 1 (mild) or 2 (moderate). Overall, two participants had Grade > 3 (severe) treatment-related TEAEs: one had worsening neutropenia and worsening thrombocytopenia, and one had worsening thrombocytopenia. One participant in the Monotherapy Dose Level 2 cohort experienced a dose limiting toxicity (DLT) of an increase in Hgb >2 g / dL, meeting protocol criteria for dose reduction at the end of Cycle 1 . There were no associated adverse events (AEs) and the maximum observed Hgb remained within normal limits. Three participants had unrelated TEAEs leading to death (transformation to AML, cerebrovascular accident (both at Dose Level 1 in combination arm), and pneumonia (Dose Level 3 in monotherapy arm)).

[0365] Changes in markers of erythropoiesis were assessed in non-TD participants to reduce the confounding effect of transfusions. Sustained increases in Hgb were observed over the first 12 weeks of treatment in non-TD participants in both the monotherapy and combination arms (FIG. 2A). Increases in sTfR, reticulocytes, and Hgb were also observed and generally were higher with increasing dose levels up through 3.0 mg / kg, supportive of increases in erythropoiesis (FIG. 2B).

[0366] Most non-TD participants in both monotherapy (FIG. 3A) and combination (FIG. 3B) arms at dose levels from 0.75 to 3.0 mg / kg achieved a mean increase in Hgb over 12 weeks. The observed increases in Hgb in the monotherapy arm suggest potential for ActRIIA / B-hFc to address anemia due to underlying MF. The observed increases in Hgb in the combination arm additionally suggest potential for ActRIIA / B- hFc to address ruxolitinib-associated anemia. Notably, increases in Hgb were achieved across the 3 lowest dose levels evaluated.

[0367] Reductions in transfusion burden over a 12-week period were assessed in TD participants based on IWG 2013 criteria and more broadly in participants receiving > 3 RBC units per 12 weeks at baseline (N=21 , 9 in Monotherapy, 12 in Combination). Reductions in transfusion burden were observed in 15 / 21 (71%) participants: nine participants (two in Monotherapy, seven in Combination) experienced a reduction of > 50%, with two participants receiving Monotherapy also achieving transfusion independence (FIGS. 4A-4B). Five TD participants in the combination arm achieved >50% reduction in transfusions. Reductions in transfusion burden were observed at the three lowest dose levels evaluated, ranging from 0.75 to 3.0 mg / kg, and occurred in participants who received up to 15 RBC units per 12 weeks at baseline. Collectively, these data support potential for ActRIIA / B-hFc to improve anemia due to MF and ruxolitinib- associated anemia, even among participants with more severe transfusion burden and greater bone marrow dysfunction.

[0368] Platelet counts were assessed in TD and non-TD participants with data pooled across dose cohorts within each of the monotherapy and combination arms (FIGS. 5A-5B). Overall, platelet counts were generally maintained or increased during the initial 12 weeks of dosing, supportive of the predicted mechanism of ActRIIA / B-hFc to promote differentiation of megakaryocytic precursors and address ineffective hematopoiesis beyond anemia.

[0369] FIG. 6 shows a case study of a participant who achieved trifactor (anemia, spleen, and symptom) improvements. The participant was a 60-year-old female with Intermediate-1 DIPSS Post-PV MF x 8 years, no response to prior ruxolitinib therapy, CALR mutation, and MF-2 to MF-3 bone marrow fibrosis who received ActRIIA / B-hFc monotherapy at 0.75 mg / kg Q4W. The participant had a Baseline transfusion burden of 5 RBC units / 12 weeks, Hgb 10.1 g / dL and achieved transfusion independence for >52 weeks with a maximum mean Hgb increase over 12 weeks of 1 .3 g / dL. The participant had marked splenomegaly at baseline with spleen volume of 2569cm3and achieved a spleen response with spleen volume reduction >35% at Week 24 (1592cm3, 38% decrease from baseline) which was generally maintained at Week 48 (1774cm3, 31 % decrease from baseline). The participant had moderate baseline symptoms with MF-SAF-TSS score of 56 and achieved a TSS response with >50% reduction with score of 25 at Week 24 which increased transiently, then fell again to 26 at Week 52. Reductions in MF-SAF- TSS were driven by improvements in itching, abdominal discomfort, fullness, and pain under left ribs. Platelet counts were maintained or increased and observed increases in sTfR and concomitant decreases in ferritin suggest improved iron utilization through erythropoiesis.

[0370] In summary, initial data support the potential for ActRIIA / B-hFc to address multiple aspects of MF, as demonstrated by observed improvements in hematopoiesis, spleen volume, and disease symptoms. For hematopoiesis, there were observed increases in markers of hematopoiesis, mean increases in hemoglobin and reduction in transfusion burden observed over 12 weeks, and maintenance or improvement in platelet counts observed. For spleen size, there was an observed reduction in spleen size in 4 / 7 (57%) evaluable participants (defined as participants with a baseline spleen size >450 cm3and a Week 24 spleen assessment) (1 / 3 monotherapy, 3 / 4 combination therapy) at Week 24, with a median reduction (n=4) of -27% (range: -47.5% to -1 1 .2%) and a median change (n=7) of -11 .2% (range: -47.5% to 30%). For symptoms, there was an observed reduction in disease symptoms in 8 / 12 (67%) evaluable participants (defined as participants with at least two symptoms with an average score > 3 or an average total score of > 10 on the MF-SAF TSS questionnaire at baseline and with a Week 24 MF-SAF-TSS assessment) at Week 24, with a median reduction (n=8) of -16.8% (range: -55.6% to -6.7%) and a median change (n=12) of -13.2% (range: -55.6% to 54.5%). These data support the potential for ActRIIA / B-hFc to ameliorate ineffective hematopoiesis and address cytopenias (anemia and thrombocytopenia) due to MF and associated with ruxolitinib, as demonstrated by data showing increases in markers of erythropoiesis, improved Hgb levels, reduced transfusion burden, and maintained or improved platelet counts, and suggest that ActRIIA / B-hFc can provide broader clinical benefit in participants with MF by decreasing spleen size and improving symptom scores.

[0371] Example 2 - Effect of ActRIIA / B-hFc in patients with anemia and myelofibrosis

[0372] A second assessment was performed on the subjects from the study described in Example 1 once additional participants had been enrolled and study duration had increased. As of this date, Part 1 dose escalation was complete and the recommended Part 2 dose (RP2D) had been identified as 3.75 mg / kg ActRIIA / B-hFc once every 4 weeks with the option to up-titrate to 5 mg / kg. At the time of the second assessment a total of 54 participants had been enrolled in both Parts 1 and 2. Key eligibility criteria:

[0373] • Transfusion dependent (TD): average of > 6 RBC units / 12 weeks at baseline with >1 transfusion within 28 days prior to treatment

[0374] • Non-transfusion dependent (Non-TD): baseline hemoglobin < 10 g / dL, with or without transfusions

[0375] • Baseline platelet count > 25 x 109 / L

[0376] As of the second assessment date, the median age of participants enrolled was 72 and the majority were male. In both treatment arms, the majority of participants had intermediate-2 to high-risk disease with more than 35% of participants in the combination arm being classified as high risk. Most participants had the JAK2 driver mutation. Interestingly, in the monotherapy arm, less than half of participants had received prior JAK inhibitor therapy. The baseline characteristics for participants in the study are provided in Table 27 below.

[0377] Table 27. Baseline Characteristics of Study Participants

[0378] CALR=calreticulin; DIPSS=dynamic international prognostic scoring system; JAK2=Janus kinase 2;

[0379] MPL=myeloproliferative leukemia protein gene

[0380] The trial enrolled a population with high disease burden. The study population was anemic and heavily transfused with approximately 1 / 3 of participants in both arms being transfusion dependent per IWG 2013 criteria and about 2 / 3 receiving at least three red blood cell units per 12 weeks at baseline. Thrombocytopenia was also prevalent, with median platelet count in both arms falling below the lower limit of normal. Thrombocytopenia was more severe in the monotherapy arm where nearly 40% had platelet levels < 50 x 109 / L. Initial exploration suggests that this more severe thrombocytopenia was mostly driven by participants previously treated with JAK inhibitors. Additionally, the majority of participants had an enlarged spleen and were symptomatic with approximately 76% of participants overall meeting criteria to be evaluable for spleen (spleen volume > 450 cm3) and symptom (based on total MF- SAF-TSS > 10***) response, including participants receiving ruxolitinib. Notably, the majority of participants in the combination arm had an enlarged spleen and symptoms indicating room for improvement beyond ruxolitinib. A summary of core disease features is provided in Table 28, below. Table 28. Core Disease Features

[0381] Hgb=hemoglobin; JAK2=Janus kinase 2; MF-SAF-TSS=myelofibrosis-symptom assessment form- total symptom score (version 4.0, 7-item); RBC = red blood cell; TD=transfusion dependent; U=units; wks=weeks

[0382] *Transfusion-dependent is based on IWG 2013 criteria (Teferri et al. Blood. 2013) and is defined as receiving > 6 RBC units in the 12 weeks prior to first dose with at least one transfusion event in the 4 weeks preceding first dose.

[0383] **Percentage based on participants with non-missing baseline value.

[0384] ***Three additional participants (one monotherapy, two combination) met criteria for being symptom response evaluable based on having at least two symptoms with an average score > 3.

[0385] As of the second assessment date, median exposure to ActRIIA / B-hFc was approximately 24 weeks in both arms, with the majority of exposure being in Part 1 participants and a median of approximately seven weeks exposure in Part 2. ActRIIA / B-hFc was generally well tolerated in participants with MF both as monotherapy and in combination with ruxolitinib. The most frequent treatment emergent adverse events in at least 15% of participants in both arms include: thrombocytopenia (10, 18.5%) (monotherapy: 7, 30.4%; combination: 3, 9.7%) and diarrhea (9 ,16.7%) (monotherapy 3, 13%; combination: 5, 16.1%). In Part 1 dose escalation, one participant (monotherapy arm, 1.5 mg / kg dose) experienced a dose limiting toxicity (DLT) of Hgb increase > 2 g / dL, which met protocol criteria for dose reduction and was not associated with AEs. There were no TESAEs considered related to ActRIIA / B-hFc by the investigator and only one TESAE of external ear neoplasm considered possibly related to ruxolitinib. There were four fatal TEAEs (pneumonia aspiration, multiple organ dysfunction, transformation to AML, cerebrovascular accident), all considered unrelated to study treatment, including one transformation to AML. Table 29 shows an overview of exposure and safety data.

[0386] Table 29. Overview of Exposure and Safety Data

[0387] TEAE = treatment-emergent adverse event; TESAE = treatment-emergent serious adverse event

[0388] *As of the second assessment date, 12 / 13 (92%) of Part 2 participants were ongoing, median exposure of 7.5 and 7.1 weeks for monotherapy and combination arms, respectively

[0389] In Part 1 , to evaluate whether there were any dose-related effects of ActRIIA / B-hFc on markers of erythropoiesis, soluble transferrin receptor, reticulocytes, and hemoglobin were assessed in nontransfusion dependent participants, to limit the confound of ongoing transfusions, with participants in each arm combined for each dose cohort. In general, increases in markers of erythropoiesis were observed that were greater at higher dose levels, supporting the activity of ActRIIA / B-hFc in driving erythropoiesis in participants with MF and anemia (FIG. 7). Variability of dose effects on hemoglobin may have been impacted by differences in transfusion status and general heterogeneity of participants across dose cohorts.

[0390] Hemoglobin responses observed with ActRIIA / B-hFc treatment are shown using waterfall plots, which show the maximum mean change in hemoglobin over any 12-week period within the first 24 weeks of treatment for non-transfusion dependent subjects treated with ActRIIA / B-hFc. Monotherapy is shown in FIG. 8A and in combination with ruxolitinib in FIG. 8B. Overall, increases in Hgb were observed in the majority of evaluable (non-TD, baseline Hgb < 10 g / dL and 12 consecutive weeks of post-baseline Hgb values) non-TD participants over a 12-week period within the first 24 weeks across both arms and across dose levels, including doses lower than the RP2D. Ten participants had an increase > 1 .0 g / dL, four had an increase > 1 .5 g / dL, and one had an increase > 2 g / dL. The observed increases in hemoglobin in the monotherapy arm suggest the ability of ActRIIA / B-hFc to address anemia due to underlying myelofibrosis, while increases in hemoglobin in the combination arm indicate the ability of ActRIIA / B-hFc to mitigate ruxolitinib-associated anemia.

[0391] The maximum reduction in transfusion burden over any 12-week period during the first 24 weeks was assessed for study participants who had received at least 3 units of red blood cells per 12 weeks at baseline, so this includes all of the transfusion-dependent participants and some of those who were not transfusion-dependent per IWG 2013 criteria. The percent change in transfusion burden for participants receiving ActRIIA / B-hFc as monotherapy is shown in FIG. 9A and for participants receiving combination therapy in FIG. 9B. Overall, most participants (20 / 33, 61%) showed some reduction in transfusion burden over the first 24 weeks of treatment. Twelve had reduction > 50% and eight achieved transfusion independence (Tl). Reductions in transfusion burden were observed in both arms and across dose levels, including doses lower than the RP2D. Notably among participants receiving 3 mg / kg of ActRIIA / B-hFc or higher in combination with ruxolitinib, approximately 73% (8 / 11) achieved at least a 50% reduction in transfusion burden and nearly half (5 / 11) achieved TL These data further support the ability of ActRIIA / B- hFc to improve anemia due to MF and ruxolitinib-associated anemia.

[0392] Ruxolitinib has been associated with dose-related thrombocytopenia that limits its clinical utility in patients with MF. In this study, the maximum mean change in platelet count over any 12 week period over the first 24 weeks was assessed in participants who were thrombocytopenic at baseline and had at least 12 weeks of post-baseline platelet data. Some improvements were observed in participants with baseline thrombocytopenia, particularly in the combination arm despite ongoing ruxolitinib treatment (FIGS. 10A- 10B). Overall, platelet count was generally maintained or improved in both monotherapy and combination arms. These data support the ability of ActRIIA / B-hFc to address MF and ruxolitinib-associated anemia while also preserving multi-lineage hematopoiesis.

[0393] Reductions in spleen size were also observed with ActRIIA / B-hFc treatment (FIGS. 11 A-11 B). Overall, more than half of participants (9 / 17, 53%) evaluable for spleen response (evaluable participants had baseline spleen size > 450 cm3and a Week 24 spleen volume assessment) showed some reduction in spleen size at Week 24, with three participants, two in the combination arm and one in the monotherapy arm, showing reductions of at least 35%. Notably, among the seven participants who had some reduction in spleen size with combination therapy, nearly all (6 / 7) had no increase in ruxolitinib dose. Reductions were observed in both arms and across dose levels, including at doses lower than RP2D. Together, the data suggest the ability of ActRIIA / B-hFc to reduce spleen size in participants with splenomegaly (i.e., improve splenomegaly), particularly among those treated with combination therapy.

[0394] Improved symptom scores were also observed with ActRIIA / B-hFc treatment (FIGS. 12A-12B). Improvements were observed in both arms and across dose levels, including at doses lower than RP2D. Overall, most evaluable participants (evaluable participants had MF-SAF-TSS > 10 or had at least two symptoms with an average score > 3 at Baseline and a Week 24 assessment) showed some improvement in symptoms at Week 24 based on reductions in MF-SAF-TSS total score. Three participants had reductions of at least 50% at Week 24, two in the monotherapy arm and one in combination with ruxolitinib.

[0395] FIGS. 13A-13B show two case studies, one from each treatment arm, that illustrate the ability of ActRIIA / B-hFc to provide multi-factor benefit. FIG. 13A shows data from a 60-year-old female with post- polycythemia-vera myelofibrosis for eight years and no response to prior ruxolitinib therapy who received ActRIIA / B-hFc monotherapy at 0.75 mg / kg, the lowest dose level studied, and achieved a trifactor response highlighted by: transfusion independence for nearly 2 years while maintaining hemoglobin of approximately 10 g / dL, platelet levels that were generally maintained or improved, spleen volume reduction of more than 35% at Week 24 which was generally maintained over nearly 2 years, and a more than 50% reduction in total symptom score that was also generally maintained over 2 years. The line at nearly two years indicates a dose increase to 1 .5 mg / kg ActRIIA / B-hFc and a corresponding increase in hemoglobin and platelets observed with this dose increase. FIG. 13B shows data from a 45-year-old female with primary MF and a ruxolitinib dose of 10 mg / day treated with 4.5 mg / kg ActRIIA / B-hFc. This participant has shown improvement across multiple parameters with the addition of ActRIIA / B-hFc, including sustained increase in hemoglobin and platelets despite ongoing ruxolitinib treatment, reduction in spleen size by approximately 25% at week 24, and 40% reduction in symptom score maintained over Weeks 8 through 32.

[0396] The ability of ActRIIA / B-hFc to address ineffective hematopoiesis in MF is supported by observed increases in hemoglobin, reduction in transfusion, and preservation or improvement of platelet counts. The results also indicate that ActRIIA / B-hFc improves splenomegaly and symptom score, particularly in combination with ruxolitinib. In summary, improvements in hemoglobin, transfusion burden, spleen volume, and total symptom scores were observed in both monotherapy and combination arms, including at dose levels below the RP2D.

[0397] Example 3 - Effect of ActRIIA / B-hFc in patients with anemia and myelofibrosis

[0398] A third assessment was performed on the subjects from the study described in Examples 1 and 2 once additional participants had been enrolled and study duration had increased. At the time of the second assessment, Part 2 was open and enrolling (32 participants enrolled, N=8 monotherapy, N=24 combination therapy) and a total of 73 participants (N=29 monotherapy, N=44 combination therapy) had been enrolled in the study. Key eligibility criteria:

[0399] • Transfusion dependent (TD): > 6 RBC units in the 12 weeks prior to first dose with >1 transfusion in the four weeks preceding first dose

[0400] • Non-transfusion dependent (Non-TD): baseline hemoglobin < 10 g / dL, with or without transfusions, but not meeting TD criteria

[0401] • Baseline platelet count > 25 x 109 / L

[0402] As of the third assessment date, the median ActRIIA / B-hFc treatment duration was 19.1 weeks (range 5 to 141 weeks) for subjects receiving monotherapy (N=29) and 24.1 weeks (range 1 to 104 weeks) for subjects receiving combination therapy (N=44). There were 11 / 29 (37.9%) subjects in the monotherapy group and 20 / 44 (45.5%) subjects in the combination therapy group who received > 6 months of treatment, and 10 / 29 (34.5%) subjects in the monotherapy group and 33 / 44 (75%) subjects in the combination therapy group with ongoing treatment. The analysis populations included 73 subjects in the safety population (received > 1 dose of ActRIIA / B-hFc), 20 subjects in the spleen response evaluable population (baseline spleen volume > 450cm3with a Week 24 assessment), 27 subjects in the symptom response evaluable population (baseline total MF-SAF-TSS > 10 or average > 3 on two items with a Week 24 assessment), 47 subjects in the anemia response evaluable population (TD or non-TD with > 12 consecutive weeks of post-baseline RBC transfusion or hemoglobin data within the first 24 weeks of treatment) for a TD vs non-TD analysis (1X1=18 TD subjects and N=29 non-TD subjects), and 57 subjects in the anemia response evaluable population for a TD3 (subjects who received > 3 RBC units in the 12 weeks prior to first dose, N=41 ) vs non-TD3 (subjects not meeting TD3 criteria with baseline hemoglobin < 10 g / dL) analysis. The baseline characteristics for participants in the study are provided in Table 30 below.

[0403] Table 30. Baseline Characteristics of Study Participants a Triple-negative was defined as the absence of JAK2, MPL, and CALR but when clinical, morphologic criteria were met and other mutation(s) was / were present, including ASXL1 , EZH2, and SRSF2. b One participant in the combination therapy arm was missing data on prior JAK inhibitor use and therefore not counted in the number with prior JAK inhibitor treatment. Per protocol the participant was receiving ruxolitinib for >8 weeks prior to start of study treatment.

[0404] CALR=calreticulin; DIPSS=dynamic international prognostic scoring system; JAK2=Janus kinase 2; MPL=myeloproliferative leukemia protein gene. The trial enrolled a population with high disease burden. The study population was anemic and heavily transfused with approximately 70% of participants receiving at least three red blood cell units per 12 weeks at baseline. Thrombocytopenia was also prevalent, with more severe thrombocytopenia in the monotherapy arm. Additionally, 63% of participants had a Baseline spleen volume > 450 cm3, including 66% of participants receiving ruxolitinib, and 73% of participants had meaningful Baseline symptom scores, including participants receiving ruxolitinib. A summary of core disease features is provided in Table 31 , below.

[0405] Table 31. Core Disease Features

[0406] Hgb = hemoglobin; MF-SAF-TSS = myelofibrosis-symptom assessment form-total symptom score (version 4.0, 7-item); RBC = red blood cell; TD = transfusion dependent; U=units.

[0407] *Transfusion-dependent (TD) is based on IWG 2013 criteria (Teferri et al., Blood, 2013) and is defined as receiving > 6 RBC units in the 12 weeks prior to first dose with at least one transfusion event in the 4 weeks preceding first dose.

[0408] **TD3 includes participants who received > 3 RBC units in the 12 weeks prior to first dose.

[0409] ActRIIA / B-hFc was generally well tolerated in participants with MF both as monotherapy and in combination with ruxolitinib. The most frequently reported treatment emergent adverse events (TEAEs) across both arms were thrombocytopenia and diarrhea. Grade >3 Thrombocytopenia occurred in 12 (16.4%) of subjects: 8 (27.4%) in the monotherapy arm and 4 (9.1 %) in the combination therapy arm. Fourteen of the fifteen participants with a TEAE of thrombocytopenia had baseline platelets < 150 x 109 / L. In Part 1 dose escalation, one participant (monotherapy, 1.5 mg / kg dose) experienced a dose limiting toxicity (DLT) of Hgb increase >2 g / dL, which met protocol criteria for dose reduction and was not associated with AEs. There were two TESAEs (anemia and fall) considered related to ActRIIA / B-hFc, and two TESAEs (anemia and external ear neoplasm) considered related to ruxolitinib by the treating Investigator. Six participants had unrelated TEAEs leading to death (pneumonia, pneumonia aspiration, multiple organ dysfunction, transformation to AML, cerebrovascular accident, septic shock). Table 32 shows an overview of safety data.

[0410] Table 32. Overview Safety Data

[0411] AE = adverse event; N / A = not applicable; TEAE = treatment-emergent adverse event; TESAE = treatment-emergent serious adverse event

[0412] Hemoglobin responses observed with ActRIIA / B-hFc treatment are shown using waterfall plots, which show the maximum mean change in hemoglobin over any 12-week period within the first 24 weeks of treatment for non-transfusion dependent subjects treated with ActRIIA / B-hFc. Monotherapy is shown in FIG. 14A and in combination with ruxolitinib in FIG. 14B. Increases in Hgb were observed in both monotherapy and combination arms in the majority of evaluable (evaluable = non-TD, baseline Hgb < 10 g / dL and > 12 consecutive weeks of post-baseline Hgb values within the first 24 weeks of treatment; non- TD participants who did not have 12 consecutive weeks of post-baseline Hgb values due to insufficient time on treatment or due to censoring for transfusions (n=18; 9 monotherapy, 9 combination) were excluded from the analysis) non-TD participants over a 12-week period within the first 24 weeks. Fifteen (15 / 29, 51 .7%) participants had an increase > 1 .0 g / dL, six (6 / 29, 20.7%) had an increase > 1 .5 g / dL, and three (3 / 29, 10.3%) had an increase > 2 g / dL. For evaluable, non-TD participants in the combination arm treated with a starting dose of ActRIIA / B-hFc of 3 mg / kg or higher, 10 / 18 (55.6%) had an increase > 1 .0 g / dL. The observed increases in hemoglobin in both arms support the ability of ActRIIA / B-hFc to address both ruxolitinib-associated anemia and anemia due to underlying myelofibrosis. The maximum reduction in transfusion burden over any 12-week period during the first 24 weeks was assessed for study participants who had received at least 3 units of red blood cells per 12 weeks at baseline (TD3 participants). The percent change in transfusion burden for participants receiving ActRIIA / B-hFc as monotherapy is shown in FIG. 15A and for participants receiving combination therapy in FIG. 15B. Overall, in both arms, reductions in transfusion burden were observed in evaluable (evaluable = TD3 with at least 12 consecutive weeks of post-baseline RBC transfusion data in the first 24 weeks; participants without 12 consecutive weeks of transfusion data (n = 10; 6 monotherapy, 4 combination) were excluded from the analysis) TD3 participants over the first 24 weeks of treatment. Sixteen (16 / 41 , 39%) had reduction > 50% and ten (10 / 41 , 24%) achieved transfusion independence (Tl). For evaluable TD3 participants in the combination arm treated with a starting dose of 3 mg / kg of ActRIIA / B-hFc or higher, 10 / 16 (62.5%) had reduction > 50% in transfusion burden and 6 / 16 (37.5%) achieved Tl. The reductions in transfusion burden observed in both arms further support the ability of ActRIIA / B-hFc to address ruxolitinib-associated anemia as well as anemia due to underlying myelofibrosis.

[0413] The mean change from baseline over time in platelet count was assessed in participants with and without baseline thrombocytopenia. Analysis was performed on the safety population who had a baseline platelet value. Approximately 62% (18 / 29) of monotherapy and 64% (28 / 44) of combination therapy participants had thrombocytopenia (<150 x 109 / L) at baseline and participants treated with monotherapy had lower platelet counts at baseline than participants in the combination arm. The mean change from baseline over time in platelet for participants receiving ActRIIA / B-hFc as monotherapy is shown in FIG. 16A and for participants receiving combination therapy in FIG. 16B. Numbers along the X-axis denote the number of participants in each group with platelet values recorded at each timepoint. Platelet values measured within seven days following a platelet transfusion were censored. Overall, platelet counts were generally stable or improved in participants in both arms, including in those with thrombocytopenia at baseline. Four participants with baseline thrombocytopenia (one in monotherapy and three in combination) achieved a maximum mean increase in platelets > 30 x 109 / L over a 12-week period in the first 24 weeks.

[0414] Reductions in spleen size were also observed with ActRIIA / B-hFc treatment in both monotherapy and combination arms at Week 24 (FIGS. 17A-17B). Evaluable participants had baseline spleen volume > 450 cm3and a Week 24 spleen volume assessment. Overall, in both arms, reductions in spleen size were observed in evaluable participants at Week 24: 8 / 20 (40%) had a reduction of > 10% and 3 / 20 (15%) had a reduction > 35%. For evaluable participants in the combination arm with a starting dose of ActRIIA / B- hFc of 3 mg / kg or higher: 7 / 8 (88%) had some reduction in spleen size at Week 24, 4 / 8 (50%) had a reduction of > 10%, and 2 / 8 (25%) had a reduction of > 35%. Observed reductions in spleen volume support the ability of ActRIIA / B-hFc to treat splenomegaly, particularly in combination with ruxolitinib.

[0415] Improved symptom scores were also observed with ActRIIA / B-hFc treatment in both monotherapy and combination arms (FIGS. 18A-18B). Overall, across both arms, MF-SAF-TSS symptom scores were reduced in 18 of 27 (67%) evaluable participants at Week 24 (evaluable participants had Baseline MF-SAF-TSS > 10 or had at least two symptoms with an average score > 3 at Baseline and a Week 24 assessment). Five evaluable participants had reductions > 50%, including three in the monotherapy arm and two in the combination arm. In addition, across symptom domains, more than a third of evaluable (evaluable = symptom response evaluable participants with a non-zero score for each item at Baseline and a non-missing Week 24 assessment; note that the number of evaluable participants changes for each item) participants in the monotherapy and combination arms achieved >25% improvement in each individual item score at Week 24 (FIGS. 19A-19B). Improvements were observed in both spleen symptoms and symptoms related to underlying myelofibrosis in participants treated with ActRIIA / B-hFc both as a monotherapy and in combination with ruxolitinib.

[0416] The ability of ActRIIA / B-hFc to address ineffective hematopoiesis in MF both as a monotherapy and in combination with ruxolitinib is supported by observed increases in hemoglobin, transfusion independence or reduction, and preservation or improvement of platelet counts. The results also indicate that ActRIIA / B-hFc improves splenomegaly, particularly in combination with ruxolitinib, and improves symptom scores, including reductions in total symptom scores as well as broad improvements across individual symptom domains both as monotherapy and in combination with ruxolitinib. In summary, improvements in hemoglobin, transfusion burden, spleen volume, and symptom scores were observed in both monotherapy and combination arms.

[0417] Example 4 - Treatment of transfusion-dependent subject having a cytopenia associated with myelofibrosis by administration of an ActRII signaling inhibitor

[0418] According to the methods disclosed herein, a physician of skill in the art can treat a transfusiondependent subject, such as a human patient, with myelofibrosis (e.g., PMF, post-ET MF, and post-PV MF) and having a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) so as to increase red blood cell count, increase hemoglobin levels, increase hematocrit, decrease RBC transfusions, promote transfusion independence, reduce spleen volume, reduce osteosclerosis, reduce bone marrow fibrosis, maintain or increase platelets, improve constitutional symptoms, and / or treat the cytopenia. The method of treatment can include diagnosing or identifying a subject as a candidate for treatment by measuring hemoglobin levels (e.g., for assessing anemia). To treat the subject, a physician of skill in the art can administer to the subject a composition containing an ActRII signaling inhibitor (e.g., an activin A antibody, a myostatin antibody, an activin B antibody, a GDF-11 antibody, an ActRII antibody, or an ActRII ligand trap, such as an ActRIIA ligand trap, an ActRIIB ligand trap, or an ActRII chimera ligand trap, e.g., An ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). The composition containing the ActRII signaling inhibitor may be administered to the subject, for example, by parenteral injection (e.g., intravenous or subcutaneous injection). The ActRII signaling inhibitor is administered in a therapeutically effective amount, such as from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the ActRII signaling inhibitor is administered bimonthly, once a month, once every four weeks, once every two weeks, or at least once a week or more (e.g., 1 , 2, 3, 4, 5, 6, or 7 times a week or more). In some embodiments, the ActRII signaling inhibitor is an ActRIIA ligand trap containing an ActRIIA variant (e.g., an ActRIIA variant fused to an Fc domain monomer by way of a linker, which may be in the form of a homodimer), such as an ActRIIA ligand trap containing an ActRIIA variant of SEQ ID NO: 69 fused to an Fc domain monomer by way of a linker in the form of a homodimer (e.g., a polypeptide of SEQ ID NO: 80 in the form of a homodimer) and is administered by subcutaneous injection once every 28 days (i.e., once every four weeks) in an amount of 0.75 mg / kg to 5 mg / kg (e.g., 3.75 mg / kg or 5 mg / kg). The ActRII signaling inhibitor is administered in an amount sufficient to increase red blood cell count, increase hemoglobin levels, increase hematocrit, decrease RBC transfusions, promote transfusion independence, reduce spleen volume, reduce osteosclerosis, reduce bone marrow fibrosis, maintain or increase platelets, improve constitutional symptoms, and / or treat the cytopenia.

[0419] Following administration of the composition to a patient, a practitioner of skill in the art can monitor the patient’s improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient’s red blood cell count, hemoglobin levels, hematocrit, spleen size, or constitutional symptoms (e.g., using MAF-SAF-TSS score). A finding that the patient’s red blood cell count, hemoglobin levels, or hematocrit are increased, that the patient’s spleen size is reduced, or that the patient’s constitution symptoms are improved following administration of the composition compared to test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0420] Other Embodiments

[0421] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0422] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0423] Other embodiments are within the following claims.

Claims

1. A method of treating a transfusion-dependent subject having a myelofibrosis-associated cytopenia, comprising the step of administering to the subject a therapeutically effective amount of an ActRII signaling inhibitor.

2. A method of treating a transfusion-dependent subject having myelofibrosis, comprising the step of administering to the subject a therapeutically effective amount of an ActRII signaling inhibitor.

3. The method according to claim 2, characterized in that the subject has cytopenia.

4. The method according to any one of claims 1-3, characterized in that the subject has discontinued treatment with the JAK inhibitor.

5. The method of claim 4, wherein the subject has experienced a relapse of the disease after treatment with a JAK inhibitor.

6. The method of claim 4, wherein the subject is resistant to treatment with a JAK inhibitor.

7. The method of claim 4, wherein the subject is intolerant to treatment with a JAK inhibitor or no longer meets the risk / benefit ratio for continued treatment with a JAK inhibitor.

8. The method according to any one of claims 1-3, characterized in that the subject is not suitable for treatment with a JAK inhibitor.

9. The method according to any one of claims 1-3, characterized in that the administration of the ActRII signaling inhibitor is performed in combination with the administration of an effective amount of a cytopenia-associated myelofibrosis therapy agent.

10. The method according to claim 9, characterized in that the administration of the combination is performed after identification of the subject as having cytopenia.

11. The method according to claim 9, characterized in that the administration in combination is performed before the patient develops cytopenia.

12. A method of treating a transfusion-dependent subject receiving a cytopenia-associated myelofibrosis therapy agent, comprising the step of administering to the subject in combination an effective amount of the cytopenia-associated myelofibrosis therapy agent and an ActRII signaling inhibitor.

13. The method according to claim 12, characterized in that the subject has cytopenia.

14. The method of claim 12 or 13, wherein the subject has myelofibrosis, polycythemia vera, steroid-resistant graft-versus-host disease, or chronic graft-versus-host disease.

15. The method of any one of claims 9-14, which improves adherence to treatment with a cytopenia-associated myelofibrosis therapy agent.

16. The method according to any one of claims 9-14, allowing for resumption of treatment with a cytopenia-associated myelofibrosis therapy agent after its cessation.

17. The method according to any one of claims 9-16, allowing to increase the dose of the cytopenia-associated myelofibrosis therapy agent.

18. The method according to any one of claims 9-16, allowing for maintaining the dose of the cytopenia-associated myelofibrosis therapy agent.

19. The method according to any one of claims 9-18, which allows for increasing the duration of treatment with a cytopenia-associated myelofibrosis therapy agent.

20. The method of any one of claims 9-19, reducing episodes of cytopenia associated with treatment with a cytopenia-associated myelofibrosis therapy agent.

21. The method according to any one of paragraphs 9-20, reducing bleeding episodes.

22. The method according to any one of paragraphs 9-21, which reduces infections.

23. The method of any one of claims 9-22, which reduces interruptions or discontinuations of treatment with a cytopenia-associated myelofibrosis therapy agent.

24. The method according to any one of claims 9-23, characterized in that the cytopenia-associated myelofibrosis therapy agent is a JAK inhibitor or imetelstat.

25. The method according to any one of claims 4-8 and 24, characterized in that the JAK inhibitor is ruxolitinib, fedratinib or pacritinib.

26. The method according to any one of paragraphs 1-11 and 14-25, characterized in that the myelofibrosis is primary myelofibrosis (PMF).

27. The method according to any one of paragraphs 1-11 and 14-25, characterized in that the myelofibrosis is myelofibrosis after essential thrombocytopenia (post-ET MF).

28. The method according to any one of paragraphs 1-11 and 14-25, characterized in that the myelofibrosis is myelofibrosis after polycythemia vera (post-PV MF).

29. The method according to any one of paragraphs 1-11 and 14-28, characterized in that the myelofibrosis is intermediate-risk myelofibrosis or high-risk myelofibrosis.

30. The method according to any one of paragraphs 1-29, reducing osteosclerosis.

31. The method according to any one of paragraphs 1-30, reducing the transfusion load of a subject.

32. The method according to any one of paragraphs 1-31, facilitating transfusion independence.

33. The method according to any one of claims 1-32, reducing splenomegaly.

34. The method according to any one of claims 1-33, which reduces bone marrow fibrosis.

35. The method according to any one of paragraphs 1-34, improving constitutional symptoms.

36. The method according to any one of paragraphs 1, 3-11 and 13-35, characterized in that the cytopenia is anemia.

37. The method according to any one of paragraphs 1, 3-11 and 13-36, characterized in that the cytopenia is thrombocytopenia.

38. The method according to any one of paragraphs 1, 3-11 and 13-37, characterized in that the cytopenia is neutropenia.

39. The method according to any one of claims 1-38, characterized in that the ActRII signaling inhibitor is an antibody against activin A or an antigen-binding fragment thereof.

40. The method according to claim 39, characterized in that the antibody against activin A is garetosmab.

41. The method according to any one of claims 1-38, characterized in that the ActRII signaling inhibitor is an antibody against myostatin or an antigen-binding fragment thereof.

42. The method according to claim 41, characterized in that the antibody against myostatin is domagrosumab, landogrosumab, alarmumab, or SRK-015.

43. The method according to any one of claims 1-38, characterized in that the inhibitor of ActRII signaling is an anti-ActRII antibody or an antigen-binding fragment thereof.

44. The method of claim 43, wherein the anti-ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861.

45. The method according to any one of claims 1-38, characterized in that the ActRII signaling inhibitor is an ActRII ligand trap.

46. ​​The method according to claim 45, characterized in that the ActRII ligand trap is an ActRIIA ligand trap.

47. The method according to claim 46, characterized in that the ActRIIA ligand trap is a composition according to Table 20.

48. The method according to claim 47, characterized in that the ActRIIA ligand trap is a polypeptide comprising a variant of extracellular ActRIIA having a sequence according to SEQ ID NO:

69.

49. The method of claim 46, wherein the ActRIIA ligand trap is sotatercept.

50. The method according to claim 45, characterized in that the ActRII ligand trap is an ActRIIB ligand trap.

51. The method of claim 50, wherein the ActRIIB ligand trap is BIIB110, ALG-802, luspatercept, ramatercept, or ACE-2494.

52. The method according to claim 50, characterized in that the ActRIIB ligand trap is a composition according to Table 21.

53. The method according to claim 45, characterized in that the ActRII ligand trap is an ActRII chimera ligand trap.

54. The method according to claim 53, characterized in that the ActRII chimera ligand trap is a composition according to Table 22 or Table 23.

55. The method according to any one of claims 1-38, characterized in that the ActRII signaling inhibitor is an antibody against activin B or an antigen-binding fragment thereof.

56. The method according to any one of claims 1-38, characterized in that the ActRII signaling inhibitor is an antibody against GDF-11 or an antigen-binding fragment thereof.