Methods of using activin receptor type ii signaling inhibitors

Abstract

The invention features methods of treating a subject having myelofibrosis by co-administering an activin receptor type II (ActRII) signaling inhibitor and a myelofibrosis therapy. The ActRII signaling inhibitor may be an antibody that binds to an ActRII ligand, an ActRII antibody, an ActRII ligand trap, or an anti-myostatin adnectin recombinant protein.

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 February 24, 2025, is named 51184-057WO2_Sequence_Listing_2_24_25.xml and is 1 ,508,634 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. Myelofibrosis is characterized by bone marrow fibrosis, ineffective hematopoiesis, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, and shortened survival. Patients often experience multiple disease-associated and treatment-emergent cytopenias, including anemia and thrombocytopenia.

[0006] There exists a need for a new therapeutic approach to ameliorate myelofibrosis-associated cytopenias, improve spleen volume, and improve patient-reported outcomes.

[0007] SUMMARY OF THE INVENTION

[0008] The invention provides methods of co-administering an activin receptor type II (ActRII) signaling inhibitor and a myelofibrosis therapy, which can be used to treat myelofibrosis or polycythemia vera or to treat a cytopenia in a subject having either of these conditions. These methods can also improve (e.g., reduce) spleen volume and improve patient-reported outcomes in a subject having myelofibrosis. The ActRII signaling inhibitor can be an antibody that binds to an ActRII ligand, an anti-ActRI I antibody, an ActRII ligand trap, or an anti-myostatin adnectin recombinant protein. Myelofibrosis therapies include momelotinib (e.g., OJJAARA), pelabresib, hydroxyurea, selinexor, gandotinib, NS-01 , TQ0510, jaktinib, flonoltinib maleate, 9-ING-41 , zilurgisertib, rineterkib, parsaclisib, umbralisib, roginolisib, TL-895, TP- 3654, thalidomide, lenalidomide, interferon (e.g., interferon alfa, peginterferon alfa-2b, ropeginterferon alfa-2b), elotuzumab, canakinumab, azacitidine, decitabine, givinostat, navitoclax, BMS-986158, zoledronic acid, danazol, navtemadlin, crizanlizumab, siremadlin, sabatolimab, NIS793, bomedemstat, DISC-0974, GB2064, PXS-5505, and reparixin.

[0009] Exemplary embodiments of the invention are described in the enumerated paragraphs below. E1 . A method of treating a subject having myelofibrosis, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0010] E2. A method of treating a subject with a myelofibrosis that has been identified as having a cytopenia, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor. E3. A method of treating a cytopenia in a subject diagnosed as having myelofibrosis, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0011] E4. A method of treating a subject having polycythemia vera, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0012] E5. The method of E1 or E4, wherein the myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination after the subject has been identified as having a cytopenia.

[0013] E6. The method of E1 or E4, wherein the myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination before the subject develops a cytopenia (e.g., to prevent or reduce the development of a cytopenia).

[0014] E7. A method of treating a cytopenia in a subject diagnosed as having polycythemia vera, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0015] E8. A method of treating a subject receiving treatment with a myelofibrosis therapy, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0016] E9. A method of treating a cytopenia in a subject receiving treatment with a myelofibrosis therapy, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0017] E10. A method of reducing transfusion burden in a subject treated with a myelofibrosis therapy, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0018] E11 . A method of promoting transfusion independence in a subject treated with a myelofibrosis therapy, comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

[0019] E12. The method of any one of E8-E11 , wherein the subject has myelofibrosis.

[0020] E13. The method of any one of E8-E11 , wherein the subject has polycythemia vera.

[0021] E14. The method of any one of E1 -E3 and E12, wherein the subject has intermediate- or high-risk myelofibrosis.

[0022] E15. The method of any one of E1 -E3, E12, and E14, wherein the subject has primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (post-ET MF), or post-polycythemia vera myelofibrosis (post-PV MF).

[0023] E16. The method of any one of E1 -E15, wherein the subject is transfusion-dependent.

[0024] E17. The method of any one of E1 -E15, wherein the subject is non-transfusion-dependent.

[0025] E18. The method of any one of E1 -E17, wherein the myelofibrosis treatment is not a cytopenia- associated myelofibrosis treatment (e.g., ruxolitinib, fedratinib, pacritinib, or imetelstat).

[0026] E19. The method of any one of E1 , E4-E6, E8, E10, and E11 , wherein the subject has a cytopenia.

[0027] E20. The method of any one of E1 , E4-E6, E8, E10, E11 , and E19, wherein the subject is identified as having a cytopenia prior to administration of the ActRII signaling inhibitor. E21 . The method of any one of E1 , E4-E6, E8, E10, E11 , E19, and E20, wherein the method further comprises identifying the subject as having a cytopenia prior to administration of the ActRII signaling inhibitor.

[0028] E22. The method of any one of E2, E3, E7, E9, and E19, wherein the cytopenia is anemia.

[0029] E23. The method of any one of E2, E3, E7, E9, E19, and E22, wherein the cytopenia is thrombocytopenia.

[0030] E24. The method of any one of E2, E3, E7, E9, E19, E22, and E23, wherein the cytopenia is neutropenia.

[0031] E25. The method of any one of E1 -E24, wherein the myelofibrosis therapy is a chemotherapeutic agent.

[0032] E26. The method of E25, wherein the chemotherapeutic agent is hydroxyurea, busulfan, selinexor, or cytarabine.

[0033] E27. The method of any one of E1 -E24, wherein the myelofibrosis therapy is a kinase inhibitor.

[0034] E28. The method of E27, wherein the kinase inhibitor is a JAK inhibitor.

[0035] E29. The method of E28, wherein the JAK inhibitor is momelotinib, lestaurtinib, AZD1480, gandotinib,

[0036] NS-018, BMS-911543, TQ05105, jaktinib, itacitinib, or flonoltinib maleate.

[0037] E30. The method of E29, wherein the JAK inhibitor is momelotinib (e.g., OJJAARA).

[0038] E31 . The method of E27, wherein the kinase inhibitor is an Aurora Kinase A inhibitor.

[0039] E32. The method of E31 , wherein the Aurora Kinase A inhibitor is alisertib.

[0040] E33. The method of E27, wherein the kinase inhibitor is a GSK-30 inhibitor.

[0041] E34. Th method of E33, wherein the GSK-30 inhibitor is 9-ING-41 .

[0042] E35. The method of E27, wherein the kinase inhibitor is an ALK2 inhibitor.

[0043] E36. The method of E35, wherein the GSK-30 inhibitor is zilurgisertib.

[0044] E37. The method of E27, wherein the kinase inhibitor is an mTOR inhibitor.

[0045] E38. The method of E37, wherein the mTOR inhibitor is everolimus.

[0046] E39. The method of E27, wherein the kinase inhibitor is an ERK1 / 2 inhibitor.

[0047] E40. The method of E39, wherein the ERK1 / 2 inhibitor is rineterkib.

[0048] E41 . The method of E27, wherein the kinase inhibitor is a PI3K inhibitor.

[0049] E42. The method of E41 , wherein the PI3K inhibitor is parsaclisib, buparlisib, umbralisib, or roginolisib.

[0050] E43. The method of E27, wherein the kinase inhibitor is TL-895, fostamatinib, rigosertib, dasatinib, TP-

[0051] 3654, PIM447, or ribociclib.

[0052] E44. The method of any one of E1 -E24, wherein the myelofibrosis therapy is an immunotherapy.

[0053] E45. The method of E44, wherein the immunotherapy is thalidomide, lenalidomide, pomalidomide, an interferon (e.g., interferon alfa, peginterferon alfa-2b, or ropeginterferon alfa-2b), elotuzumab, or canakinumab.

[0054] E46. The method of any one of E1 -E24, wherein the myelofibrosis therapy is a hypomethylating agent.

[0055] E47. The method of E46, wherein the hypomethylating agent is azacitidine or decitabine.

[0056] E48. The method of any one of E1 -E24, wherein the myelofibrosis therapy is a histone deacetylase

[0057] (HDAC) inhibitor.

[0058] E49. The method of E48, wherein the HDAC inhibitor is givinostat or panobinostat. E50. The method of any one of E1 -E24, wherein the myelofibrosis therapy is a bromodomain and extra-terminal (BET) protein inhibitor.

[0059] E51 . The method of E50, wherein the BET inhibitor is pelabresib, BMS-986158, mivebresib, or ABBV- 744.

[0060] E52. The method of E51 , wherein the BET inhibitor is pelabresib.

[0061] E53. The method of any one of E1 -E24, wherein the myelofibrosis therapy is a Hedgehog inhibitor.

[0062] E54. The method of E53, wherein the Hedgehog inhibitor is glasdegib, I PI-926, sonidegib, or vismodegib.

[0063] E55. The method of any one of E1 -E24, wherein the myelofibrosis therapy is navitoclax (ABT-263), zoledronic acid, danazol, LCL-161 , navtemadlin, crizanlizumab, siremadlin, sabatolimab, NIS793, bomedemstat, DISC-0974, zinpentraxin alfa, AVID200, tagraxofusp, pevonedistat, plitidepsin, GB2064, PXS-5505, tipifarnib, reparixin, or enasidenib.

[0064] E56. The method of E55, wherein the myelofibrosis therapy is navitoclax.

[0065] E57. The method of E55, wherein the myelofibrosis therapy is GB2064.

[0066] E58. The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is an activin A antibody or an antigen binding fragment thereof.

[0067] E59. The method of E58, wherein the activin A antibody is garetosmab.

[0068] E60. The method of E58, 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 ).

[0069] E61 . The method of E58 or E60, 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).

[0070] E62. The method of any one of E58, E60, and E61 , wherein 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 (e.g., a heavy chain and light chain sequence from the same row of Table 3).

[0071] E63. The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is a myostatin antibody or an antigen binding fragment thereof.

[0072] E64. The method of E63, wherein the myostatin antibody is domagrozumab, landogrozumab, trevogrumab, or apitegromab (SRK-015).

[0073] E65. The method of E63, 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).

[0074] E66. The method of E63 or E65, 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).

[0075] E67. The method of any one of E63, E65, and E66, 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).

[0076] E68. The method of E63, wherein the myostatin antibody or an antigen binding fragment thereof is a bi-specific antibody or an antigen binding fragment thereof that also binds to activin A.

[0077] E69. The method of E68, wherein 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 , 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 ), 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, 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).

[0078] E70. The method of E68 or E69, wherein 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).

[0079] E71 . The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is an ActRII antibody or an antigen binding fragment thereof.

[0080] E72. The method of E71 , wherein the ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861 .

[0081] E73. The method of E71 , wherein the ActRII antibody or an antigen binding fragment thereof has a

[0082] 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). E74. The method of E71 or E71 , 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).

[0083] E75. The method of any one of E71 , E73, and E74, 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 11 (e.g., a heavy chain and light chain sequence from the same row of Table 11 ).

[0084] E76. The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is an ActRII ligand trap.

[0085] E77. The method of E76, wherein the ActRII ligand trap is an ActRIIA ligand trap.

[0086] E78. The method of E77, wherein the ActRIIA ligand trap is a composition of Table 21 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 21 ).

[0087] E79. The method of E78, wherein the ActRIIA ligand trap comprises the sequence of any one of SEQ ID NOs: 6-72.

[0088] E80. The method of E79, wherein the ActRIIA ligand trap comprises the sequence of SEQ ID NO: 69.

[0089] E81 . The method of any one of E77-E80, wherein the ActRIIA ligand trap comprises an Fc domain or an Fc domain monomer.

[0090] E82. The method of E81 , wherein the ActRIIA ligand trap comprises the sequence of SEQ ID NO: 80.

[0091] E83. The method of E81 or E82, wherein the ActRIIA ligand trap is in the form of a dimer (e.g., homodimer).

[0092] E84. The method of E77, wherein the ActRIIA ligand trap comprises an extracellular portion of wildtype ActRIIA (e.g., SEQ ID NO: 73 or SEQ ID NO: 729).

[0093] E85. The method of E77, wherein the ActRIIA ligand trap is sotatercept.

[0094] E86. The method of E76, wherein the ActRII ligand trap is an ActRIIB ligand trap.

[0095] E87. The method of E86, wherein the ActRIIB ligand trap comprises an extracellular portion of wildtype ActRIIB (e.g., SEQ ID NO: 74 or a portion thereof).

[0096] E88. The method of E86, wherein the ActRIIB ligand trap is BIIB110, ALG-802, luspatercept, ramatercept, or ACE-2494.

[0097] E89. The method of E86, wherein the ActRIIB ligand trap is a composition of Table 22 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 22).

[0098] E90. The method of E89, 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).

[0099] E91 . The method of E76, wherein the ActRII ligand trap is an ActRII chimera ligand trap.

[0100] E92. The method of E91 , wherein the ActRII chimera ligand trap is a composition of Table 23 or Table

[0101] 24 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 23 or Table 24).

[0102] E93. The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is an activin B antibody or an antigen binding fragment thereof. E94. The method of E93, 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.

[0103] E95. The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is a GDF-11 antibody or an antigen binding fragment thereof.

[0104] E96. The method of any one of E1 -E57, wherein the ActRII signaling inhibitor is an anti-myostatin adnectin recombinant protein.

[0105] E97. The method of E96, wherein the anti-myostatin adnectin recombinant protein is taldefgrobep alfa.

[0106] E98. The method of E96, wherein the anti-myostatin adnectin recombinant protein comprises a 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 sequence provided in Table 20.

[0107] E99. The method of any one of E1 -E98, wherein the method further includes evaluating red cell or platelet parameters after administration of the ActRII signaling inhibitor.

[0108] E100. The method of any one of E1 -E99, 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 or pretreatment measurements).

[0109] E101 . The method of any one of E1 -E100, 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 in the 8 or 12 weeks preceding treatment).

[0110] E102. The method of E101 , wherein the method leads to a reduction in transfusion burden of at least 50% of the number of RBC units transfused (e.g., at least a 50% 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 in the 8 or 12 weeks preceding treatment).

[0111] E103. The method of any one of E1 -E102, wherein the subject achieves transfusion independence for at least twelve weeks during treatment (e.g., compared to pretreatment transfusion data from the 12 weeks directly preceding treatment).

[0112] E104. The method of any one of E1 -E103, wherein the method reduces spleen volume.

[0113] E105. The method of any one of E1 -E104, wherein the method improves patient-reported outcomes (e.g., leads to an improvement in MF-SAF-TSS, PROMIS score or BFI score from baseline).

[0114] E106. The method of any one of E1 -E105, 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, or reduce susceptibility to infection.

[0115] E107. The method of any one of E1 -E106, wherein the ActRII signaling inhibitor is administered in an amount sufficient to ameliorates a myelofibrosis-associated cytopenia, reduce spleen volume, reduce bone marrow fibrosis, improve hematopoiesis in the bone marrow, reduce osteosclerosis, improve bone marrow fibrosis grade, increase body weight, treat or reduce cachexia, slow or inhibit the progression to AML and / or accelerated MF, or improve patient-reported outcomes.

[0116] E108. The method of any one of E1 -E107, wherein the method does not cause a vascular complication in the subject.

[0117] E109. The method of E108, wherein the method does not increase vascular permeability or leakage. E110. The method of any one of E1 -E109, wherein the subject is a human.

[0118] Definitions

[0119] 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.

[0120] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0121] 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.

[0122] As used herein, “administration” refers to providing or giving a subject a therapeutic agent (e.g., an ActRII signaling inhibitor and a myelofibrosis therapy described herein), by any effective route. Exemplary routes of administration are described herein below.

[0123] 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.

[0124] “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.

[0125] 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.

[0126] Wild-type, human ActRIIA precursor protein (SEQ ID NO: 75):

[0127] / WGZW / WAF / FL / SCSSGAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFAT WKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPT SNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPL QLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRG TSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHR DIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRID MYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKH AGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL

[0128] 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).

[0129] As used herein, the terms “extracellular activin receptor type IIB (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: 1046 shown below). The sequence of the wild-type, human ActRIIB is shown below (SEQ ID NO: 1046), in which the signal peptide is italicized and the extracellular portion is bold.

[0130] Wild-type human ActRIIB (SEQ ID NO: 1046):

[0131] MTAPWV’A / . / .WGS / .CAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCY ASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAG GPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPP PSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMK HENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSY LHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVG TRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQ HPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSL IRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI 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.

[0132] 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: 1046 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: 3) and wild-type, human ActRIIA (SEQ ID NO: 1 ) 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 1015-1045. In particular embodiments, an extracellular ActRII chimera has the sequence of any one of SEQ ID NOs: 772-793 (Table 17).

[0133] 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.

[0134] 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 -744)) 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 1015-1045 (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).

[0135] 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). Exemplary cytopenia-associated myelofibrosis treatments include ruxolitinib (e.g., JAKAFI® / JAKAVI®), fedratinib (e.g., INREBIC®), pacritinib (e.g., VONJO™), and imetelstat.

[0136] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) fused to a moiety. The moiety may increase stability or improve pharmacokinetic properties of the polypeptide. The moiety (e.g., an Fc domain monomer, an 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.

[0137] 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., 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.

[0138] 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).

[0139] 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.

[0140] 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 ).

[0141] 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).

[0142] 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, 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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, in an ActRII ligand trap described herein, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof 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 portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof 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.

[0143] As used herein, the term “C-terminal extension” refers to the addition of one or more amino acids to the C-terminus of 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 to the C-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (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.

[0144] 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:

[0145] 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. 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.

[0146] 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.

[0147] 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 kOff / kOn.

[0148] 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 an ActRII signaling inhibitor and a myelofibrosis therapy 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, spleen volume, 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] As used herein, the term “myelofibrosis therapy” refers to an agent that is FDA approved for the treatment of subjects with myelofibrosis, that is used to treat subjects with myelofibrosis (including off- label use), that is currently in clinical trials for the treatment of subjects with myelofibrosis, or that has been evaluated in clinical trials for the treatment of subjects with myelofibrosis. As used herein, the term “myelofibrosis therapy” does not include antibodies that bind to an ActRII ligand, anti-ActRI I antibodies, ActRII ligand traps (e.g., sotatercept, luspatercept, or an ActRIIA ligand trap including the sequence of SEQ ID NO: 69), or anti-myostatin adnectin recombinant proteins. 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.

[0157] 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.

[0158] 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 portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof 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.

[0159] 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 portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may form a heterodimer through the interaction of two Fc domain monomers, each fused to a different extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof, which form an Fc domain in the heterodimer.

[0160] 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).

[0161] As used herein, the terms “effective amount” and “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector described herein 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 myelofibrosis, 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.

[0162] 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.

[0163] 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.

[0164] 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).

[0165] 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.

[0166] 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.

[0167] 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.

[0168] DETAILED DESCRIPTION OF THE INVENTION

[0169] The invention features methods of co-administering an activin receptor type II (ActRII) signaling inhibitor and a myelofibrosis therapy. The ActRII signaling inhibitor can be an antibody that binds to an ActRII ligand, an anti-ActRI I antibody, an ActRII ligand trap, or an anti-myostatin adnectin recombinant protein, and exemplary myelofibrosis therapies include momelotinib (e.g., OJJAARA), pelabresib, hydroxyurea, selinexor, gandotinib, NS-01 , TQ0510, jaktinib, flonoltinib maleate, 9-ING-41 , zilurgisertib, rineterkib, parsaclisib, umbralisib, roginolisib, TL-895, TP-3654, thalidomide, lenalidomide, interferon (e.g., interferon alfa, peginterferon alfa-2b, ropeginterferon alfa-2b), elotuzumab, canakinumab, azacitidine, decitabine, givinostat, navitoclax, BMS-986158, zoledronic acid, danazol, navtemadlin, crizanlizumab, siremadlin, sabatolimab, NIS793, bomedemstat, DISC-0974, GB2064, PXS-5505, and reparixin. These methods can be used to treat myelofibrosis, such as intermediate (e.g., intermediate-1 or intermediate -2) or high-risk myelofibrosis, including primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF). These methods can also reduce or ameliorate cytopenias (e.g., anemia, thrombocytopenia, and / or neutropenia), improve (e.g., reduce) spleen volume, and improve patient-reported outcomes.

[0170] ActRII signaling

[0171] 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).

[0172] 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. Relatedly, 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. These data suggest that increased signaling through endogenous activin receptors, either due to increased expression of activin receptor ligands (e.g., activin A, activin B, myostatin) or increased expression of activin receptors themselves, could disrupt hematopoiesis. Methods that reduce or inhibit activin A, activin B, and / or myostatin signaling could, therefore, be used to promote hematopoiesis and treat diseases and conditions involving ineffective hematopoiesis, such as a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis. ActRII signaling inhibitors

[0173] 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.

[0174] Activin A antibodies

[0175] 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, and 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.

[0176] 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, 831 , 833, 835, 837, 838, 840, 842, 843, 845, 847, 849, 851 , 853, 854, 856, 858, and 860, 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, 830, 832, 834, 836, 839, 841 , 844, 846, 848, 850, 852, 855, 857, and 859. 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 .

[0177] Table 1. Exemplary HCVR and LCVR sequences of activin A antibodies

[0178] 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, 864, 870, 882, 901 , 912, 923, and 929; 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, 865, 874, 883, 892, 896, 902, 907, 913, 924, and 930; 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, 866, 871 , 875, 877, 884, 887, 893, 897, 903, 908, 914, 919, 925, and 931 ; 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, 861 , 867, 872, 876, 878, 880, 885, 889, 894, 898, 904, 909, 916, 920, 926, and 932; 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, 862, 868, 873, 879, 881 , 886, 888, 890, 895, 899, 905, 910, 915, 917, 921 , 927, and 933; 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, 863, 869, 891 , 900, 906, 911 , 918, 922, and 928. 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.

[0179] Table 2. Exemplary CDR sequences of activin A antibodies

[0180] 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: 934 and 935; 936 and 937; 938 and 939; 940 and 941 ; 942 and 935; 943 and 944; 945 and 946; 947 and 946; 948 and 949; 950 and 951 ; 952 and 953; 954 and 955; 956 and 957; 958 and 957; 959 and 960; 961 and 962; 963 and 964; or 965 and 966 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%,

[0181] 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

[0182] Myostatin antibodies

[0183] 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.

[0184] 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, 969, 977, 983, 985, 987, 989, 991 , 993, 995, 997, 999, 1001 , and 1003, 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, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 299, 307, 309, 311 , 313, 315, 317, 319, 321 , 358, 372, 374, 388, 390, 392, 406, 408, 410, 412, 414, 416, 418, 420, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 443, 445, 447, 477-486, 970, 978, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 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.

[0185] Table 4. Exemplary HCVR and LCVR sequences of myostatin antibodies

[0186]

[0187] 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, 971 , 974, 975, 1006, and 1010; 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, 972, 976, and 1007; 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, 979, and 1008; 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, 967, 980, and 101 1 ; 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, 973, 981 , 1009, and 1012; 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, 968, and 1005. 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 SEQ ID NO: 343; 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 SEQ ID NO: 347; 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 SEQ ID NO: 350; 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 SEQ ID NO: 332; 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 SEQ ID NO: 335; 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 SEQ ID NO: 337. 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.

[0188] Table 5. Exemplary CDR sequences of myostatin antibodies

[0189]

[0190] Table 6. Exemplary heavy chain CDR sequences of myostatin antibodies

[0191] Table 7. Exemplary light chain CDR sequences of myostatin antibodies

[0192] 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; 369 and 370, or 1013 and 1014 (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).

[0193] Table 8. Exemplary heavy and light chain sequences of myostatin antibodies

[0194] 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, 174, 831 , 833, 835, 837, 838, 840, 842, 843, 845, 847, 849, 851 , 853, 854, 856, 858, and 860, 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, 830, 832, 834, 836, 839, 841 , 844, 846, 848, 850, 852, 855, 857, and 859) 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, 448-476, 969, 977, 983, 985, 987, 989, 991 , 993, 995, 997, 999, 1001 , and 1003, 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, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 299, 307, 309, 311 , 313, 315, 317, 319, 321 , 358, 372, 374, 388, 390, 392, 406, 408, 410, 412, 414, 416, 418, 420, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 443, 445, 447, 477-486, 970, 978, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, and 1004). 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 .

[0195] Activin B antibodies

[0196] 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.

[0197] MKCSWIMFFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMYWVK

[0198] QRPGQGLEWIGMIHPNSGSTNYNGKFKTGATLTVDKSSSTVYMQLSSLTSEDSAVYYC

[0199] ARWGYGGNYDYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSL (SEQ ID NO: 494)

[0200] MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQ QKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISTMEAEDAVTYYCHQYHRSP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK (SEQ ID NO: 495)

[0201] In some embodiments, the ActRII signaling inhibitor is a GDF-11 antibody or an antigen binding fragment thereof.

[0202] ActRII antibodies

[0203] 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.

[0204] 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. Table 9. Exemplary HCVR and LCVR sequences of ActRII antibodies

[0205] 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, 71 1 , 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%, 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.

[0206] Table 10. Exemplary CDR sequences of ActRII antibodies

[0207] 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 11 . 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

[0208] 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

[0209]

[0210] ActRII ligand traps

[0211] 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.

[0212] 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.

[0213] Human ActRIIA, extracellular portion (SEQ ID NO: 73):

[0214] GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS

[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), 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), increasing megakaryocyte progenitors, 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 -1 1 , 1 -9, 1 -7, 1 -5, 1 -3, or 1 -2; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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 described herein. To maintain polypeptide function, it is important that the lysine (K) at position X17 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 described herein, 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 described herein 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)) described herein to provide reduced BMP9 binding.

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

[0222] The ActRIIA variants described herein 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 described herein 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 described herein 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)).

[0223] In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X3 is E, Xe is R, Xn is D, X12 is K, X13 is R, X 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; X 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; X is K or R; X 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; Xn is D; X12 is K; X13 is R; X14 is L; X15 is F or Y; X16 is K or R; X17 is K; X 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.

[0224] 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).

[0225] Table 13. Extracellular ActRIIA variants having the sequences of SEQ ID NOs: 6-72 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.

[0226] 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).

[0227] 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 of the ActRIIA variant). 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 of the ActRIIA variant (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids).

[0228] 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.

[0229] 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.

[0230] 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-1 1 ) 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, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher).

[0231] In some embodiments, the ActRIIA ligand trap is sotatercept (also known as ACE-01 1 ). Additional ActRIIA ligand traps that may be used in the methods described herein include those 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, 1 1 ,013,785, and 7,951 ,771 , each of which is incorporated herein by reference.

[0232] 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.

[0233] Human ActRIIB, extracellular portion (SEQ ID NO: 74):

[0234] GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWL DDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT

[0235] 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 BIIB1 10 (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. WQ2010062383, WO2015192127, WQ2019140283, and WQ2021 189010; US Patent Application Publication Nos. US201 10250198 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, 10,358,633, 1 1 ,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, 9,273,1 14, and 10,308,704, each of which is incorporated herein by reference. In some embodiments, the ActRIIB ligand trap contains an ActRIIB variant having the sequence of SEQ ID NO: 730 shown in Table 14.

[0236] Table 14. Amino acid substitutions in an extracellular ActRIIB variant having a sequence of SEQ ID NO: 730

[0237] In some embodiments, the ActRIIB variant has the sequence of any one of SEQ ID NOs: 731 -744 (Table 15).

[0238] 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. Accordingly, in some embodiments, the N-terminal portion of ActRIIB begins with the amino acid in the fifth position of SEQ ID NO: 74 (A), while in other embodiments (e.g., in embodiments in which the six amino acids found at the N-terminus of extracellular ActRIIA are not included in the chimera), the N-terminal portion of ActRIIB begins with the amino acid in the first position of SEQ ID NO: 74 (G). 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, in which case the N-terminal portion of ActRIIB begins with the amino acid in the ninth position of SEQ ID NO: 74. 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 US 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 GDF1 1 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 X3, X4, X5, and Xe. In some embodiments, BMP9 binding is reduced in extracellular ActRII chimeras containing the amino acid sequence KKDS (SEQ ID NO: 1047) or TKDS (SEQ ID NO: 1048) at positions X3, X4, X5, and 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 TEEN (SEQ ID NO: 76) at positions X3, X4, X5, and Xe can have a substitution of the amino acid K for the amino acid E at position X4. 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 X3, X4, X5, and Xe can have a substitution of the amino acid E for the amino acid K at position X4. 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 GAILGRAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2

[0251] X3CYDRQECVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 757)

[0252] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRRHCFATWKNISGSIEIVKQGCWLDDX2X3C

[0253] YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 758)

[0254] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCFATWKNISGSIEIVKQGCWLDDX2X3C

[0255] YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 759)

[0256] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWKNISGSIEIVKQGCWLDDX2X3C

[0257] YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 760)

[0258] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGSIEIVKQGCWLDDX2X3C

[0259] YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 761 )

[0260] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIEIVKQGCWLDDX2X3C

[0261] YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 762)

[0262] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2X3

[0263] CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 763)

[0264] GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2X3

[0265] CYDRQECVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 764)

[0266] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRRHCFATWKNISGSIEIVKQGCWLDDX2X3

[0267] CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 765)

[0268] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCFATWKNISGSIEIVKQGCWLDDX2X3

[0269] CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 766)

[0270] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWKNISGSIEIVKQGCWLDDX2X3 CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 767)

[0271] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGSIEIVKQGCWLDDX2

[0272] X3CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 768)

[0273] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIEIVKQGCWLDDX2

[0274] X3CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 769)

[0275] GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2

[0276] 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, Xs is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 751 -771 , 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, Xs is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 751 -771 , Xi is D, X2 is F, X3 is N, X4 is E, X5 is K, Xe is K, X7 is D, Xs is S, and X9 is Q.

[0277] In some embodiments, ActRII chimera ligand trap contains the sequence of any one of SEQ ID NOs: 772-793 (Table 17).

[0278] Table 17. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 772-793

[0279] 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

[0280] 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 1 1 (GDF-1 1 ) 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, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher).

[0281] 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 (PSA) can be replaced with the 5thp-sheet from ActRIIB (PSB)). Each ActRII protein has seven p- sheets (pi -p?) and eight intervening sequences (Xi-Xs). The ActRII chimeras include at least one of pia, p2a, p3a, p4a, psa, or p7a and at least one of p , p2b, p3b, p4b, psb, 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, ps, 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, Xs, 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: 81 1 ), which is a portion of TIELVKKGCW (p4b) (SEQ ID NO: 810); at least VE, which is a portion of VEK (psa); at least V, which is a portion of VAT (psb); 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. WO2022235620 and WO2024102906, the disclosures of which are incorporated herein by reference.

[0282] Table 18. Extracellular ActRII chimera sequences

[0283] In some embodiments, the ActRII chimera has the sequence of an ActRII chimera listed in Table , below. Table 19. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 1015-1045

[0284] 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).

[0285] 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).

[0286] 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 described herein 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.

[0287] 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.

[0288] Anti-myostatin adnectin recombinant proteins

[0289] In some embodiments, the ActRII signaling inhibitor is an anti-myostatin adnectin recombinant protein. Anti-myostatin adnectin recombinant proteins bind specifically to myostatin and reduce myostatin signaling.

[0290] Anti-myostatin adnectin recombinant proteins in clinical trials include taldefgrobep alfa (Phase 3). Taldefgrobep alfa (also known as BMS-986089, RG6206, RO7239361 , and BHV-2000) is anti-myostatin adnectin recombinant protein dimer having the sequence of SEQ ID NO: 1051 that binds to the C- terminus of mature myostatin and to the ActRIIB-myostatin complex. Taldefgrobep alfa is described in International Patent Application Publication No. WO2014043344 and Muntoni et al. (Neurol. Ther. 13(1 ) :183-219, 2024), each of which is incorporated herein by reference.

[0291] Additional anti-myostatin adnectin recombinant proteins that may be used in the methods described herein include those described in International Patent Application Publication No. WO2014043344, which is incorporated herein by reference. In some embodiments, the anti-myostatin adnectin recombinant protein includes a sequence listed in Table 20. In some embodiments, the anti- myostatin adnectin recombinant protein includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 20, such as any one of SEQ ID NOs: 1051 -1137. In some embodiments, the anti-myostatin adnectin recombinant protein includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 20 from which the His6 tag has been removed (e.g., the anti-myostatin adnectin recombinant protein includes a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1053-1108 from which the C-terminal His6 tag has been removed). In some embodiments, the anti-myostatin adnectin recombinant protein has a sequence provided in Table 20. In some embodiments, the anti-myostatin adnectin recombinant protein includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 20 fused to an Fc domain by way of a linker (e.g., a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1052-1108 and 1130-1137 fused to an Fc domain). In some embodiments, an anti-myostatin adnectin recombinant protein of Table 20 fused to an Fc domain forms a dimer (e.g., a homodimer or heterodimer, such as a dimer of any one of SEQ ID NOs: 1051 and 1109-1129, e.g., a homodimer of SEQ ID NO: 1051 ). Table 20. Exemplary anti-myostatin adnectin recombinant proteins

[0292] Myelofibrosis therapies

[0293] An ActRII signaling inhibitor described herein can be administered to a subject in combination with a myelofibrosis therapy. The subject may already be undergoing treatment with the myelofibrosis therapy when treatment with the ActRII signaling inhibitor is initiated or the subject may begin treatment with both agents concurrently.

[0294] Chemotherapy

[0295] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with a chemotherapy for myelofibrosis. There are multiple chemotherapeutic agents that have been used for or that are being developed for the treatment of myelofibrosis, including hydroxyurea, busulfan, selinexor, and cytarabine.

[0296] Hydroxyurea (also known as hydroxycarbamide) is a non-alkylating antineoplastic agent used for cytoreduction in myeloproliferative neoplasms. It is frequently used to attenuate hyperproliferative manifestations related to PMF. Hydroxyurea can be administered orally (e.g., in the form of a capsule). Hydroxyurea may be administered once a day or once every third day.

[0297] Busulfan is an alkylating agent that has been administered to subjects in combination with fludarabine for the treatment of myelofibrosis. Busulfan can be administered via intravenous infusion, e.g., at a dose of 0.8 mg / kg body weight.

[0298] Selinexor is a nuclear export inhibitor that is currently being evaluated in a Phase 2 clinical trial for the treatment of myelofibrosis in subjects who previously received treatment with a JAK inhibitor. Selinexor can be administered orally, e.g., in the form of a tablet, such as at a dose of 60 mg or 80 mg once weekly.

[0299] Cytarabine (cytosine arabinoside, ara-C) is an antimetabolite evaluated in an early Phase 1 clinical trial for the treatment of subjects with myelofibrosis in combination with arsenic trioxide.

[0300] Cytarabine can be administered by injection, e.g., subcutaneous injection, such as at a dose of 10 mg / m2twice daily.

[0301] Kinase inhibitors

[0302] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with a kinase inhibitor, such as a JAK inhibitor. Momelotinib is a kinase inhibitor that was recently approved for the treatment of myelofibrosis (under brand name OJJAARA) and there are many additional kinase inhibitors in clinical trials for treating myelofibrosis, including JAK inhibitors, an Aurora Kinase inhibitor, an ERK inhibitor, PIM kinase inhibitors, and PI3K inhibitors, among others.

[0303] Momelotinib is a kinase inhibitor (JAK1 / JAK2 / AVCR1 inhibitor) FDA approved for the treatment of intermediate or high-risk myelofibrosis (MF), including primary MF or secondary MF (post-polycythemia vera (PV) and post-essential thrombocythemia (ET)), in adults with anemia (under brand name OJJAARA). It can be administered orally in the form of a tablet. Recommended dosing is 200 mg orally once daily, which can be reduced to 150 mg orally once daily for subjects with hepatic impairment.

[0304] Lestaurtinib (CEP-701 ) is a JAK2 inhibitor evaluated in a Phase 2 clinical trial for the treatment of myelofibrosis (chronic idiopathic myelofibrosis). Lestaurtinib can be administered orally, such as at a dose of 80 mg orally twice a day.

[0305] AZD1480 is a JAK2 inhibitor evaluated in a Phase 1 / 2 clinical trial for the treatment of myelofibrosis (PMF, post- PV MF, or post-ET MF). AZD1480 can be orally administered, e.g., in the form of a capsule.

[0306] Gandotinib (LY2784544) is a JAK2 inhibitor in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis. Gandotinib can be administered orally, such as at a dose of 120 mg once daily.

[0307] NS-018 is a JAK2 inhibitor in Phase 2 clinical trials for the treatment of subjects with myelofibrosis (PMF, post- PV MF, or post-ET MF). NS-018 can be administered orally, such as at a dose of 75-400 mg once daily or 100-400 mg twice daily, such as 300 mg once or twice daily.

[0308] BMS-911543 is a JAK2 inhibitor evaluated in a Phase 1 / 2 clinical trial for the treatment of subjects with symptomatic intermediate-1 , intermediate-2, or high-risk myelofibrosis. BMS-911543 can be administered orally, e.g., in the form of a capsule, such as at 10-200 mg twice daily.

[0309] TQ05105 is a JAK1 / 2 and ROCK 1 / 2 inhibitor currently Phase 2 clinical trials for the treatment of subjects with myelofibrosis, such as intermediate- and high-risk myelofibrosis. TQ05105 can be administered orally, e.g., in the form of a tablet.

[0310] Jaktinib is a JAK and ACVR1 inhibitor in Phase 3 clinical trials for the treatment of subjects with myelofibrosis (e.g., intermediate-2 or high-risk myelofibrosis). Jaktinib can be administered orally, e.g., in the form of a tablet, such as at a dose of 100 mg twice daily.

[0311] Itacitinib is a JAK1 inhibitor that was evaluated in Phase 2 clinical trials for the treatment of subjects with myelofibrosis (subjects with PMF, post-PV-MF, or post-ET-MF who received prior ruxolitinib and / or fedratinib monotherapy). Itacitinib can be administered orally, such as at a dose of 200 mg or 600 mg twice daily.

[0312] Flonoltinib maleate is a JAK2 / FLT3 inhibitor in Phase 1 / 2 clinical trials for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF and medium-risk-1 or high risk). Flonoltinib maleate can be administered orally, e.g., in the form of a tablet, such as once daily at a dose of 25 mg- 325 mg.

[0313] Alisertib is an Aurora Kinase A inhibitor that was evaluated in a Phase 1 clinical trial for the treatment of subjects having myelofibrosis (PMF, post-PV-MF, or post-ET-MF; intermediate-1 risk or beyond by the Dynamic International Prognostic Scoring System (DIPSS); and in need of treatment, intolerant or refractory to ruxolitinib (or other investigational JAK inhibitors), or unlikely to benefit from ruxolitinib). Alisertib can be administered orally, e.g., at a dose of 50 mg twice daily for days 1 -7 of a 21 - day cycle.

[0314] 9-ING-41 is a small molecule, maleimide-based, selective GSK-30 inhibitor in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF or post-ET-MF) alone or in combination with ruxolitinib. 9-ING-41 can be administered by injection, e.g., by intravenous infusion, such as at a dose of 9.3 mg / kg twice weekly. Zilurgisertib (INCB000928) is a small molecule ALK2 inhibitor in a Phase 1 / 2 clinical study for the treatment of subjects with myelofibrosis and anemia alone or in combination with ruxolitinib. Zilurgisertib can be administered orally, e.g., once daily at a dose of 50 mg or 100 mg.

[0315] Everolimus (RAD001 ) is a small molecule mTOR inhibitor that was evaluated in a Phase 1 / 2 clinical trial for treatment of subjects with myelofibrosis. Everolimus can be administered orally, such as at a dose of 5 mg, 7.5 mg, or 10 mg daily.

[0316] Rineterkib (LTT462) is an ERK1 / 2 inhibitor that is in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Rineterkib can be administered orally, e.g., in the form of a capsule.

[0317] Parsaclisib (INCB050465) is a PI3K5 inhibitor in Phase 3 clinical trials for the treatment of subjects with myelofibrosis in combination with ruxolitinib. Parsaclisib can be administered orally, e.g., once per day.

[0318] Buparlisib (BKM120) is a PI3K inhibitor evaluated in a Phase 1 clinical trial for the treatment of subjects with myelofibrosis (primary MF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Buparlisib can be administered orally, e.g., in the form of a capsule, such as once per day.

[0319] Umbralisib (TGR-1202) is a PI3K5 inhibitor in a Phase 1 clinical trial for the treatment of subjects with myeloproliferative neoplasms (polycythemia vera, PMF, post-PV MF, or post-ET MF) in combination with ruxolitinib, Umbralisib can be administered orally, e.g., in the form of a tablet.

[0320] IOA-244 (Roginolisib) is a PI3K5 inhibitor in a Phase 1 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV MF, or post-ET-MF) in combination with ruxolitinib. IOA-244 can be administered orally, such as once per day.

[0321] TL-895 is a highly selective irreversible tyrosine kinase inhibitor in a Phase 2 clinical trial for the treatment of myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in subjects who are relapsed / refractory, intolerant, or ineligible to receive JAK inhibitor treatment. TL-895 can be administered orally, for example, once or twice daily at a dose of 150, 300, or 450 mg.

[0322] Fostamatinib is a spleen tyrosine kinase (Syk) inhibitor evaluated in a Phase 2 clinical trial for the treatment of myelofibrosis (PMF, post-PV MF, or post-ET MF and high-risk, intermediate-2 risk, or intermediate-1 risk by IPSS) in subjects with severe thrombocytopenia. Fostamatinib can be administered orally, e.g., in the form of a tablet, such as at a dose of 100 or 150 mg twice daily.

[0323] LNK01002 is a triple kinase inhibitor evaluated in a Phase 1 clinical trial in subjects with myelofibrosis (PMF, post-PV MF, or post-ET MF). LNK01002 can be administered orally, such as at a dose of 15-260 mg twice daily.

[0324] Rigosertib is a multi-kinase inhibitor evaluated in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV MF, or post-ET MF) and anemia. Rigosertib can be administered orally, e.g., in the form of a capsule, such as at a dose of 560 mg twice a day.

[0325] Dasatinib is a tyrosine kinase inhibitor evaluated in a Phase 2 clinical trial for treatment of subjects with myelofibrosis (PMF). Dasatinib can be administered orally, e.g., in the form of a tablet, such as at a dose of 70 mg twice daily.

[0326] TP-3654 is a PIM1 kinase inhibitor in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (intermediate or high-risk primary or secondary MF) who are relapsed, refractory, intolerant to, or not candidates for JAK inhibitors. TP-3654 can be administered orally, such as at a dose of 480-720 mg once daily or 360-1440 mg twice daily.

[0327] PIM447 (LGH447) is a pan-PIM kinase inhibitor evaluated in a Phase 1 clinical trial for treatment of subjects with myelofibrosis (JAK2V617F-positive primary or secondary MF) in combination with ruxolitinib or both ruxolitinib and LEE011 . PIM447 can be administered orally, such as at a dose of 70-700 mg once daily.

[0328] LEE011 (Ribociclib) is a CDK4 / 6 inhibitor evaluated in a Phase 1 clinical trial for treatment of subjects with myelofibrosis (JAK2V617F-positive primary or secondary MF) in combination with ruxolitinib or both ruxolitinib and PIM447. LEE01 lean be administered orally.

[0329] Immunotherapy

[0330] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with an immunotherapy. Exemplary immunotherapies that have been used for or are being evaluated for the treatment of myelofibrosis include thalidomide and its analogs, interferons, elotuzumab, and canakinumab.

[0331] Thalidomide is an antiangiogenic and immunomodulatory drug in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (either primary or post-ET / PV requiring therapy, including those previously treated and relapsed or refractory, or if newly diagnosed, with intermediate-1 or -2 or high risk) in combination with ruxolitinib. Thalidomide can be administered orally, e.g., in the form of a capsule, and may be administered once per day. Thalidomide analogs lenalidomide and pomalidomide have also been evaluated in clinical trials (Phase 2 and Phase 3, respectively) for the treatment of myelofibrosis. Lenalidomide can be administered orally, e.g., in the form of a capsule, such as at a dose of 5 mg, 10 mg, or 25 mg once daily. Pomalidomide can be administered orally, e.g., in the form of a capsule, such as at a dose of 0.5 mg once daily.

[0332] Interferon alpha (IFN-a) has been used in various formulations and dosages to treat myelofibrosis and is currently recommended as an alternative to ruxolitinib or hydroxyurea for the treatment of low-risk myelofibrosis by the National Comprehensive Cancer Network. Different formulations of IFN-a have been evaluated for treatment of myelofibrosis (interferon alfa and peginterferon alfa-2b (Phase 3)) and ropeginterferon alfa-2b has been used off-label in subjects with myelofibrosis (FDA approved for treatment of polycythemia vera) and is currently in Phase 2 clinical trials for treating subjects with myelofibrosis (under the name P1101 (polyethyleneglycol [PEG]-proline-interferon alpha-2b)). Formulations of IFN-a (e.g., interferon alfa, peginterferon alfa-2b, or ropeginterferon alfa-2b) can be administered by injection, e.g., subcutaneous injection.

[0333] Elotuzumab is a is a SLAMF7-directed immunostimulatory monoclonal antibody that is in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF and intermediate or higher risk disease). Elotuzumab can be administered intravenously, e.g., by intravenous injection or intravenous infusion, such as by intravenous injection or infusion once per week.

[0334] Canakinumab is monoclonal antibody targeting interleukin-1 p that is in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF). Canakinumab can be administered by injection, e.g., subcutaneous injection, such as at a dose of 200 mg every three weeks. Hypomethylating agents

[0335] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with a hypomethylating agent. Hypomethylating agents that are currently being studied in clinical trials for the treatment of subjects with myelofibrosis include azacitidine and decitabine.

[0336] Azacitidine is a nucleoside metabolic inhibitor that is in a Phase 2 clinical trial for treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. It has also been evaluated in prior Phase 2 clinical trials as a monotherapy for the treatment of myelofibrosis. Azacitidine can be administered by injection, e.g., subcutaneous or intravenous infusion, such as daily for 4 or 5 days per 28-day cycle.

[0337] Decitabine is a nucleoside metabolic inhibitor in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (all subtypes in subjects with anemia or splenomegaly). Decitabine can be administered by injection, e.g., subcutaneous injection or intravenous infusion, such as daily subcutaneous injection on days 1 -5 and 8-12 of a 42-day cycle.

[0338] Histone deacetylase inhibitors

[0339] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with a histone deacetylase (HDAC) inhibitor. HDAC inhibitors that are currently in clinical trials for treating myelofibrosis or that have been studied in clinical trials for treating myelofibrosis include givinostat and panobinostat.

[0340] Givinostat (ITF2357) is an HDAC inhibitor in Phase 2 clinical trials for treating subjects with polycythemia vera, essential thrombocythemia, primary MF, post-PV MF, and post-ET MF. Givinostat can be administered orally, e.g., in the form of a capsule, such as at a dose of 50 mg, 75 mg, or 100 mg once, twice, or three times per day.

[0341] Panobinostat is an HDAC inhibitor that was evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) and found to reduce spleen volume. Panobinostat can be administered orally, e.g., in the form of a capsule, such as at a dose of 25 mg or 40 mg thrice weekly.

[0342] BET inhibitors

[0343] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with a bromodomain and extra-terminal (BET) protein inhibitor. BET inhibitors that have been or are currently in clinical trials for the treatment of subjects with myelofibrosis include pelabresib, BMS-986158, mivebresib, and ABBV-744.

[0344] Pelabresib (CPI-0610)is a small molecule BET inhibitor that is being evaluated in a Phase 3 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Pelabresib can be administered orally, such as at a dose of 125-175 mg once daily for 14 days, followed by a seven-day break in a 21 -day cycle.

[0345] BMS-986158 is a small molecule BET inhibitor that is being evaluated in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) as a monotherapy or in combination with ruxolitinib or fedratinib. BMS-986158 can be administered orally, such as at a dose of 0.75-4.5 mg at a schedule of five days on and 2 days off. Mivebresib (ABBV-075) is a small molecule BET inhibitor that was evaluated in a Phase 1 clinical trial for the treatment of subjects with myelofibrosis (intermediate- or high-risk PMF, post-PV-MF, or post- ET-MF) alone or in combination with ruxolitinib or navitoclax. Mivebresib can be administered orally, e.g., in the form of a tablet.

[0346] ABBV-744 is a small molecule BET inhibitor that was evaluated in a Phase 1 clinical trial for the treatment of subjects in myelofibrosis (intermediate- or high-risk PMF, post-PV-MF, or post-ET-MF) alone or in combination with ruxolitinib or navitoclax. ABBV-744 can be administered orally, e.g., in the form of a tablet.

[0347] Hedgehog inhibitors

[0348] In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject in combination with a hedgehog inhibitor. Hedgehog inhibitors that have been or are currently in clinical trials for the treatment of subjects with myelofibrosis include glasdegib, I PI-926, LDE225, and Vismodegib.

[0349] Glasdegib (PF-04449913) is a small molecule inhibitor of the Hedgehog pathway evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) previously treated with one or more JAK inhibitors. Glasdegib can be administered orally, e.g., in the form of a tablet, such as at a dose of 100 mg daily.

[0350] I PI-926 is a small molecule inhibitor of the Hedgehog pathway evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (primary or secondary (post-PV or post-ET) MF). I PI-926 can be administered orally, such as once daily at a dose of 160 mg.

[0351] LDE225 (Sonidegib) is a small molecule inhibitor of the Hedgehog signaling pathway evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. LDE225 can be administered orally, e.g., in the form of a capsule, such as at a dose of 400 mg daily.

[0352] Vismodegib is a small molecule inhibitor of the Hedgehog pathway evaluated in a Phase 1 clinical trial for treating subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF and intermediate-1 , intermediate-2, or high-risk by DIPSS) in combination with ruxolitinib. Vismodegib can be administered orally, e.g., in the form of a capsule, such as at a dose of 150 mg once daily.

[0353] Other agents

[0354] Many other agents with differing mechanisms of action have been or are currently being evaluated for the treatment of subjects with myelofibrosis. In some embodiments, an ActRII signaling inhibitor described herein is administered to a subject with myelofibrosis in combination with one of these agents, such as an agent listed below.

[0355] Zoledronic acid is a bisphosphonate that can be used to relieve bone pain in subjects with myelofibrosis. It was also evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis. Zoledronic acid can be administered by injection (e.g., intravenous infusion) and may be administered once a week, once every three weeks, or once every four weeks.

[0356] Danazol is a synthetic steroid derived from ethisterone that was used to treat subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in a Phase 3 clinical trial. Danazol treatment results in symptom and spleen improvements but does not address anemia. Danazol can be administered orally, e.g., in the form of a capsule, such as at a dose of 300 mg twice a day.

[0357] LCL-161 is a second mitochondrial-derived activator of caspases (SMAC) mimetic evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF). LCL-161 can be administered orally, such as at a dose of 1500 mg once per week.

[0358] Navtemadlin (KRT-232) is small molecule inhibitor of murine double minute 2 (MDM2) that is in a Phase 2 / 3 trial for the treatment of subjects with myelofibrosis who no longer benefit from treatment with a JAK inhibitor. Navtemadlin can be administered orally, such as at a dose of 120 mg or 240 mg once daily for five or seven days in a 21 - or 28-day cycle.

[0359] Crizanlizumab (SEG101 , SelG-1 ) is a monoclonal antibody that binds to p-selectin that is in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Crizanlizumab can be administered by injection, e.g., intravenous infusion.

[0360] Siremadlin (HDM-201 ) is a p53-MDM2 inhibitor that is in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Siremadlin can be administered orally, e.g., in the form of a capsule, such as at a dose of 10 mg, 20, or 40 mg once daily on days 1 -5 of a 28-day cycle.

[0361] Sabatolimab (MBG453) is a monoclonal antibody that binds to TIM-3 that is in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Sabatolimab can be administered by injection, e.g., intravenous infusion.

[0362] NIS793 is an anti-TGF-p monoclonal antibody that is in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. NIS793 can be administered by injection, e.g., intravenous infusion.

[0363] Bomedemstat (IMG-7289) is a small molecule lysine-specific demethylase 1 (LSD1 ) inhibitor that is in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET- MF) in combination with ruxolitinib. Bomedemstat can be administered orally, e.g., in the form of a capsule, such as at a dose of 0.4 mg / kg once daily.

[0364] DISC-0974 is an anti-hemojuvelin antibody that is in a Phase 1 / 2 clinical trial for treatment of subjects with myelofibrosis (PMF, post-PV MF, or post-ET MF with intermediate-2 or high-risk MF by DIPSS) and anemia. DISC-0974 can be administered by injection, e.g., subcutaneous injection, such as at a frequency of once every four weeks.

[0365] Zinpentraxin alfa (PRM-151 , RO7490677) is a recombinant human pentraxin 2 protein evaluated in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET- MF) alone or in combination with ruxolitinib. Zinpentraxin alfa can be administered by injection e.g., by intravenous infusion, such as at a dose of 0.3 mg / kg, 3 mg / kg, or 10 mg / kg for 3-5 days in a 28-day cycle.

[0366] AVID200 is a potent and selective TGFp 1 / 3 trap evaluated in Phase 1 clinical trial in subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF and intermediate-2 or high-risk disease). AVID200 can be administered by injection, e.g., intravenous infusion, such as at a dose of 70 mg / m2or 180 mg / m2on day 1 of a 21 -day cycle.

[0367] Tagraxofusp (SL-401 ) is a CD123-directed cytotoxin (IL-3 fused to diphtheria toxin) evaluated in a Phase 2 clinical trial for treatment of subjects with myelofibrosis (intermediate-2 or high-risk MF) who are resistant / refractory or intolerant to approved JAK therapy. Tagraxofusp can be administered by injection, e.g., as an intravenous infusion, such as at 12 mcg / kg daily on days 1 -3 of a 21 -, 28-, or 42-day cycle.

[0368] Pevonedistat (TAK-924 / MLN4924) is a Nedd8-activating enzyme inhibitor evaluated in a Phase 1 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF and intermediate-1 , intermediate-2, or high-risk by IPSS) in combination with ruxolitinib, Pevonedistat can be administered intravenously, e.g., via IV infusion, such as at a dose of 5, 10, or 20 mg / m2 on days 1 , 3, and 5 of a 28-day cycle.

[0369] Plitidepsin is a didemnin evaluated in a Phase 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV MF, or post-ET MF), Plitidepsin can be administered intravenously, e.g., via IV infusion, such as at a dose of 5 mg / m2 on days 1 and 15 of a 28-day cycle.

[0370] GB2064 (PAT-1251 ) is a lysyl oxidase-like-2 (LOXL-2) inhibitor in a Phase 3 clinical trial for the treatment of subjects with myelofibrosis (PMF or secondary MF). GB2064 can be administered orally, e.g., in the form of a tablet, such as at a dose of 4 x 250 mg tablets twice daily.

[0371] PXS-5505 is a pan-lysyl oxidase (LOX) inhibitor in a Phase 1 / 2 clinical trial for the treatment of subjects with myelofibrosis (PMF, post-PV MF, or post-ET MF). PXS-5505 can be administered orally, e.g., in the form of a capsule, such as at a dose of 100 mg twice daily.

[0372] Tipifarnib is a farnesyltransferase inhibitor evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (PMF, post-PV MF, or post-ET MF). Tipifarnib can be administered orally, such as at a dose of 300 mg twice daily for 21 days in a 28-day cycle.

[0373] Reparixin is a CXCR1 and CXCR2 inhibitor in a Phase 2 clinical trial for treating subjects with myelofibrosis (intermediate-2 or high-risk PMF, post-ET MF, or post-PV MF). Reparixin can be administered orally, such as at a dose of 1200 mg three times daily.

[0374] Enasidenib is an IDH2 inhibitor evaluated in a Phase 2 clinical trial for treating subjects with myelofibrosis (MF with an IDH2 mutation) in combination with ruxolitinib. Enasidenib can be administered orally, such as at a dose of 50-100 mg daily.

[0375] Navitoclax (ABT-263) is a B-cell lymphoma (BCL)-2, BCL-XL, and BCL-W inhibitor that is being evaluated in Phase 3 clinical trials for the treatment of subjects with myelofibrosis (PMF, post-PV-MF, or post-ET-MF) in combination with ruxolitinib. Navitoclax can be administered orally, e.g., as a tablet, such as at a dose of 100 mg or 200 mg once daily.

[0376] Fc domains

[0377] 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 IgG 1 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 ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is shown below (SEQ ID NO: 97):

[0378] THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0379] 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:

[0380] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0381] 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):

[0382] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0383] In some embodiments, an Fc domain 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 is from an lgG1 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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 an 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 an 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., IgG 1 , 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).

[0384] 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.

[0385] Methods of engineering Fc domains that have reduced dimerization are known in the art. In some embodiments, one or more amino acids with large side-chains (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.

[0386] 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 / 00241 1 1 , all of which are incorporated herein by reference in their entireties. In some embodiments, 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 . .

[0387] Albumin-binding peptides

[0388] 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-binding peptides can improve the pharmacokinetics of protein pharmaceuticals.

[0389] As one example, albumin-binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin binding peptide includes the sequence DICLPRWGCLW (SEQ ID NO: 83).

[0390] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) to increase the serum half-life of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof. 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.

[0391] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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 wishing to be bound by 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. Fibronectin domains

[0392] 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.

[0393] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 - 793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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.

[0394] 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 .

[0395] GPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIK GLKPGVVYEGQLISIQQYGHQEVTRFDFTTTST

[0396] In another embodiment, the fibronectin domain is an adnectin protein.

[0397] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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 wishing to be bound by 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. Serum albumin

[0398] 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.

[0399] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015- 1045 (e.g., SEQ ID NOs: 772-793)) 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.

[0400] 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).

[0401] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEP ERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVV LNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

[0402] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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 wishing to be bound by 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.

[0403] Linkers

[0404] An ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)), 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, 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 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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).

[0405] 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).

[0406] 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.

[0407] 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)), is an amino acid spacer having the sequence GGG. For example, an ActRIIA ligand trap 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 or SEQ ID NO: 84) 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):

[0408] GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKG CWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0409] In another example, a polypeptide described herein can contain an extracellular ActRIIB variant (e.g., any one of SEQ ID NOs: 730-744) fused to an Fc domain monomer (e.g., SEQ ID NO: 79 or SEQ ID NO: 84) 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: 1049): GRGEAETRECLYYNANWELERTNQSGVERCEGEKDKRLHCYASWRNSSGSLEIVKKG CWLDDFNCYDRDTCVATKENPQVYFCCCEGNMCNERFTHLPEAGGPEVTYEPPPTAP TGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0410] In another example, a polypeptide described herein can contain an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 751 -793) fused to an Fc domain monomer (e.g., SEQ ID NO: 79 or SEQ ID NO: 84) 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: 1050): GAILGRSETQECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEIVKQ GCWLDDFNCYDRTDCVEKKDSPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0411] The C-terminal Lys339 of the polypeptide of SEQ ID NO: 1050 (the C-terminal Lys in the Fc region of SEQ ID NO: 1050) and the C-terminal Lys345 of the polypeptide of SEQ ID NO: 1049 (the C-terminal Lys in the Fc region of SEQ ID NO: 1049) may or may not be present, without affecting the structure or stability of the polypeptide. The disclosure specifically contemplates SEQ ID NO: 1050 that does not include the C-terminal Lys corresponding to Lys339 and SEQ ID NO: 1049 that does not include the C- terminal Lys corresponding to Lys345. The polypeptide of SEQ ID NO: 1050 may be expressed including a C-terminal Lys339 which then may be proteolytically cleaved upon expression of the polypeptide, and the polypeptide of SEQ ID NO: 1049 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 NO: 1050 and SEQ ID NO: 1049 are expressed using nucleic acid constructs encoding the polypeptide including a C-terminal lysine residue). The polypeptides of SEQ ID NO: 1050 and SEQ ID NO: 1049 may also be expressed without including the C-terminal Lys339 and the C-terminal Lys345, respectively.

[0412] Vectors, host cells, and protein production

[0413] The ActRII signaling inhibitors described herein 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.

[0414] Nucleic acid vector construction and host cells

[0415] A nucleic acid sequence encoding the amino acid sequence of a polypeptide described herein 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, PCR mutagenesis, ligation, and overlap extension PCR. A nucleic acid molecule encoding a polypeptide described herein 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.

[0416] A nucleic acid sequence encoding a polypeptide described herein 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.

[0417] 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. co / / strains include, but are not limited to, E. coli 294 (ATCC®31 ,446), E. coli h 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.

[0418] Protein production, recovery, and purification

[0419] Host cells used to produce the polypeptides described herein 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.

[0420] 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 described herein 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.

[0421] 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).

[0422] Alternatively, the polypeptides described herein can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administering 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. 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.

[0423] Pharmaceutical compositions and preparations

[0424] The invention features pharmaceutical compositions that include the ActRII signaling inhibitors described herein, 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)). In some embodiments, a pharmaceutical composition of the invention includes an ActRII 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) or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 or 1015-1045 (e.g., SEQ ID NOs: 772-793)) 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 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), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 730-750 (e.g., SEQ ID NOs: 731 -744), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 751 -793 and 1015-1045 (e.g., SEQ ID NOs: 772-793)) fused to a moiety (e.g., an 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 including a polypeptide described herein 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 described herein, or a vector containing such a nucleic acid molecule.

[0425] 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).

[0426] The pharmaceutical compositions 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.

[0427] The pharmaceutical compositions 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 described herein. 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.

[0428] The pharmaceutical composition may be formed in a unit dose form as needed. The amount of active component, e.g., a polypeptide described herein, 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).

[0429] 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.

[0430] Routes, dosage, and administration

[0431] Pharmaceutical compositions that include the polypeptides described herein 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).

[0432] In some embodiments, a pharmaceutical composition that includes a nucleic acid molecule encoding a polypeptide described herein 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 described herein may be administered directly to a subject.

[0433] 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.

[0434] 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 may include a dosage of an ActRII signaling inhibitor described herein 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, 0.6, 0.7, 0.75, , 0.8, 0.9, 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.

[0435] 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 described herein 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 described herein 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 increase as the medical condition improves or decrease as the health of the patient declines.

[0436] Methods of treatment

[0437] 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, an ActRII ligand trap, or an anti-myostatin adnectin recombinant protein) can be used to treat a subject already receiving a myelofibrosis therapy or can be administered combination with a myelofibrosis therapy. 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). 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 myelofibrosis therapy 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 myelofibrosis is intermediate (e.g., intermediate-1 or intermediate-2) or high-risk myelofibrosis. 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 is receiving treatment with a myelofibrosis therapy for polycythemia vera. In some embodiments, the subject has anemia. Anemia can be 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 subject is transfusion-dependent. In some embodiments, the subject is non-transfusion-dependent. In some embodiments, the subject has been receiving treatment with the myelofibrosis therapy for at least eight weeks (e.g., 8 weeks or longer, such as 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 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) prior to co-administration of an ActRII signaling inhibitor described herein. In some embodiments, the subject has been receiving treatment with the myelofibrosis therapy for at least eight weeks (e.g., 8 weeks or longer, such as 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 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 myelofibrosis therapy 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, 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 myelofibrosis therapy 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 myelofibrosis therapy 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 myelofibrosis therapy 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 myelofibrosis therapy 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). In some embodiments, the subject is identified as having a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) prior to co-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 co-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).

[0438] In some embodiments, the myelofibrosis therapy is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is hydroxyurea, busulfan, selinexor, or cytarabine. In some embodiments, the myelofibrosis therapy is a kinase inhibitor, such as a JAK inhibitor, Aurora Kinase inhibitor, ERK inhibitor, PIM kinase inhibitor, or PI3K inhibitor. In some embodiments, the kinase inhibitor is a JAK inhibitor, such as momelotinib, lestaurtinib (CEP-701 ), AZD1480, gandotinib (LY2784544), NS- 018, BMS-911543, TQ05105, jaktinib, itacitinib, or flonoltinib maleate. In some embodiments, the kinase inhibitor is momelotinib (e.g., OJJAARA). In some embodiments, the kinase inhibitor is alisertib (an Aurora Kinase A inhibitor), 9-ING-41 (a GSK-30 inhibitor), zilurgisertib (INCB000928, an ALK2 inhibitor), everolimus (RAD001 , an mTOR inhibitor), rineterkib (LTT462, an ERK1 / 2 inhibitor), TL-895 (tyrosine kinase inhibitor), fostamatinib (a spleen tyrosine kinase (Syk) inhibitor), LNK01002 (a triple kinase inhibitor), rigosertib (multi-kinase inhibitor), dasatinib (tyrosine kinase inhibitor, or LEE011 (ribociclib, CDK4 / 6 inhibitor). In some embodiments, the kinase inhibitor is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is parsaclisib (INCB050465), buparlisib (BKM120), umbralisib (TGR- 1202), or IOA-244 (roginolisib). In some embodiments, the kinase inhibitor is a PIM kinase inhibitor. In some embodiments, the PIM kinase inhibitor is TP-3654 or PIM447 (LGH447). In some embodiments, the myelofibrosis therapy is an immunotherapy. In some embodiments, the immunotherapy is thalidomide or an analog thereof (e.g., lenalidomide or pomalidomide), an interferon (e.g., an IFN-a formulation, e.g., interferon alfa, peginterferon alfa-2b, or ropeginterferon alfa-2b (P1101 , polyethyleneglycol [PEG]-proline- interferon alpha-2b)), elotuzumab, or canakinumab. In some embodiments, the myelofibrosis therapy is a hypomethylating agent. In some embodiments, the hypomethylating agent is azacitidine or decitabine. In some embodiments, the myelofibrosis therapy is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is givinostat (ITF2357) or panobinostat. In some embodiments, the myelofibrosis therapy is a BET inhibitor. In some embodiments, the BET inhibitor is pelabresib (CPI-0610), BMS-986158, mivebresib (ABBV-075), or ABBV-744. In some embodiments, the BET inhibitor is pelabresib. In some embodiments, the myelofibrosis therapy is a Hedgehog signaling inhibitor. In some embodiments, the hedgehog signaling inhibitor is glasdegib (PF-04449913), IPI-926, LDE225 (sonidegib), or vismodegib. In some embodiments, the myelofibrosis therapy is navitoclax (ABT-263), zoledronic acid, danazol, LCL- 161 , navtemadlin (KRT-232), crizanlizumab (SEG101 , SelG-1 ), siremadlin (HDM-201 ), sabatolimab (MBG453), NIS793, bomedemstat (IMG-7289), DISC-0974, zinpentraxin alfa (PRM-151 , RO7490677), AVID200, tagraxofusp (SL-401 ), pevonedistat (TAK-924 / MLN4924), plitidepsin, GB2064 (PAT-1251 ), PXS-5505, tipifarnib, reparixin, or enasidenib. In some embodiments, the myelofibrosis therapy is not a cytopenia-associated myelofibrosis treatment (e.g., ruxolitinib, fedratinib, pacritinib, or imetelstat).

[0439] 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 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 compared to measurements obtained prior to treatment or compared to measurements obtained from subjects treated only with a myelofibrosis therapy. 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), which may prevent or reduce inflammation / fibrosis, restore hematopoiesis in the bone marrow, and treat cytopenias due to myelofibrosis. 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 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 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) and reverse reductions in red blood cells, platelets, and / or neutrophils. In some embodiments, the methods described herein reduce bleeding events. In some embodiments, the methods described herein decrease infections.

[0440] 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 subject with lower transfusion needs at baseline (e.g., a transfusion-independent subject or a non- transfusion-dependent subject requiring 1 , 2, 3, 4, or 5 RBC transfusions at baseline) 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, for example, within the first 24 weeks or 52 weeks of treatment of a transfusion-independent 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 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, treatment according to the methods described herein reduces the number of RBC transfusions by at least 50% (e.g., by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) 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 for a subject with higher transfusion needs at baseline (e.g., a transfusion-dependent subject, such as a subject receiving 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, or more RBC transfusions at baseline). In some embodiments, the compositions and methods described herein promote transfusion independence (e.g., a subject who required 1 or more (e.g., 1 , 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 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, such as for a subject with higher transfusion needs at baseline (e.g., a transfusion-dependent subject, such as a subject receiving 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, or more RBC transfusions at baseline)). Concurrent treatment for anemia with RBC transfusions is recommended 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 or magnetic resonance imaging, 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 MF (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 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 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. 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 24 weeks or 52 weeks of treatment as described herein.

[0441] In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage.

[0442] In some embodiments the ActRII signaling inhibitor that is co-administered with a myelofibrosis therapy 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, 0.6, 0.7, 0.75, 0.8, 0.9, 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.

[0443] Compositions that can be administered to a subject according to the methods described herein are provided in Tables 21 -24, below.

[0444] Combination therapy

[0445] An ActRII signaling inhibitor described herein is to be administered to the subject in combination with a myelofibrosis therapy. The myelofibrosis therapy may be, e.g., hydroxyurea, busulfan, selinexor, cytarabine, momelotinib (e.g., OJJAARA), lestaurtinib (CEP-701 ), AZD1480, gandotinib (LY2784544), NS-018, BMS-911543, TQ05105, jaktinib, itacitinib, flonoltinib maleate, alisertib, 9-ING-41 , zilurgisertib, everolimus, rineterkib, TL-895, fostamatinib, LNK01002, rigosertib, dasatinib, LEE011 (ribociclib), parsaclisib (INCB050465), buparlisib (BKM120), umbralisib (TGR-1202), IOA-244 (roginolisib), TP-3654, PIM447 (LGH447), thalidomide, lenalidomide, pomalidomide, an interferon (e.g., interferon alfa, peginterferon alfa-2b, or ropeginterferon alfa-2b), elotuzumab, canakinumab, azacitidine, decitabine, givinostat (ITF2357), panobinostat, navitoclax (ABT-263), pelcitoclax (APG-1252), pelabresib (CPI-0610), BMS-986158, mivebresib (ABBV-075), ABBV-744,glasdegib (PF-04449913), IPI-926, LDE225 (sonidegib), vismodegib, zoledronic acid, danazol, LCL-161 , navtemadlin (KRT-232), crizanlizumab (SEG101 , SelG-1 ), siremadlin (HDM-201 ), sabatolimab (MBG453), NIS793, bomedemstat (IMG-7289), DISC-0974, zinpentraxin alfa (PRM-151 , RO7490677), AVID200, tagraxofusp (SL-401 ), pevonedistat (TAK-924 / MLN4924), plitidepsin, GB2064 (PAT-1251 ), PXS-5505, tipifarnib, reparixin, or enasidenib. The myelofibrosis therapy 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 myelofibrosis therapy 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 myelofibrosis therapy 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 myelofibrosis therapy 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 myelofibrosis therapy 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 an anti- myostatin adnectin recombinant protein) or intravenous (e.g., for an Activin A, Activin B, myostatin, GDF- 11 , or ActRII antibody) injection or infusion while the myelofibrosis therapy can be administered orally (for hydroxyurea, selinexor, momelotinib (e.g., OJJAARA), lestaurtinib, AZD1480, gandotinib, NS-018, BMS- 911543, TQ05105, jaktinib, itacitinib, flonoltinib maleate, alisertib, zilurgisertib, everolimus, rineterkib, parsaclisib, buparlisib, umbralisib, roginolisib, TL-895, fostamatinib, LNK01002, rigosertib, dasatinib, TP- 3654, PIM447, ribociclib, thalidomide, lenalidomide, pomalidomide, givinostat, panobinostat, navitoclax, pelabresib, BMS-986158, mivebresib, ABBV-744, glasdegib, IPI-926, sonidegib, vismodegib, danazol, LCL-161 , navtemadlin, siremadlin, bomedemstat, GB2064, PXS-5505, tipifarnib, reparixin, and enasidenib), by subcutaneous injection (for cytarabine, interferon alfa, peginterferon alfa-2b, ropeginterferon alfa-2b, canakinumab, azacitidine, decitabine, and DISC-0974), or by intravenous injection or infusion (for busulfan, 9-ING-41 , elotuzumab, azacitidine, decitabine, pelcitoclax, zoledronic acid, crizanlizumab, sabatolimab, NIS793, zinpentraxin alfa, AVID200, tagraxofusp, pevonedistat, and plitidepsin). 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 myelofibrosis therapy. In some embodiments, the ActRII signaling inhibitor and the myelofibrosis therapy 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 myelofibrosis therapy can be administered once, twice, or three times daily or two, three, four, or five times per week. In some embodiments, the ActRII signaling inhibitor and the myelofibrosis therapy are administered at the same or at similar frequencies. For example, both the ActRII signaling inhibitor and the myelofibrosis therapy 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.

[0446] 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, 0.6, 0.7, 0.75, 0.8, 0.9, 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. The myelofibrosis therapy can be administered as indicated on the label (e.g., for OJJAARA), as described in the clinical trial protocol for treatment of myelofibrosis, or as recommended by a physician of skill in the art.

[0447] In some embodiments, combination therapy with an ActRII signaling inhibitor and a myelofibrosis therapy as described herein ameliorates a myelofibrosis-associated cytopenia (e.g., ameliorates anemia, thrombocytopenia, and / or neutropenia associated with myelofibrosis), improves spleen volume (e.g., reduces splenomegaly or spleen volume), improves or restores hematopoiesis in the bone marrow, reduces bone marrow fibrosis or slows the progression of bone marrow fibrosis (e.g., improves bone marrow fibrosis grade from baseline or prevents bone marrow fibrosis grade from worsening), ameliorates attenuated bone resorption and osteosclerosis, increases body weight, treats or reduces cachexia, increases hemoglobin levels, reduces transfusion burden (e.g., reduces the number of RBC transfusions, such as by at least 50% compared to baseline), promotes transfusion independence, slows or inhibits the progression to AML (bone marrow blasts >20%) and / or accelerated MF (bone marrow blasts >10%), increases platelet count, increases reticulocyte count, and / or improves patient-reported outcomes (e.g., improves MF-SAF-TSS, PROMIS score or BFI score from baseline).

[0448] Kits

[0449] An ActRII signaling inhibitor and a myelofibrosis therapy 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 myelofibrosis therapy. Table 21

[0450]

[0451] Table 22

[0452] Table 23

[0453] Table 24

[0454] EXAMPLES

[0455] 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.

[0456] Example 1 - Treatment of myelofibrosis by co-administration of an ActRII signaling inhibitor and a myelofibrosis therapy

[0457] According to the methods disclosed herein, a physician of skill in the art can treat a subject, such as a human patient, diagnosed as having myelofibrosis (e.g., PMF, post-ET MF, or post-PV MF) and a cytopenia (e.g., anemia, thrombocytopenia, and / or neutropenia) so as to increase red blood cell count, increase hemoglobin levels, decrease RBC transfusions, promote transfusion independence, treat the cytopenia, improve spleen volume, and / or improve patient-reported outcomes. 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, an ActRII ligand trap, or an anti-myostatin adnectin recombinant protein, such as an ActRIIA ligand trap containing 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), such as an ActRIIA variant fused to an Fc domain or Fc domain monomer)) in combination with a composition containing a myelofibrosis therapy. The composition containing the ActRII ligand trap may be administered to the subject, for example, by parenteral injection (e.g., intravenous or subcutaneous injection) in combination with a myelofibrosis therapy, which may be administered orally, by subcutaneous injection, or by intravenous injection or infusion. The ActRII signaling inhibitor (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 an Fc domain or an Fc domain monomer) 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, 0.6, 0.7, 0.75, 0.8, 0.9, 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 extracellular 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) and the myelofibrosis therapy is momelotinib (e.g., OJJAARA) or pelabresib and is administered orally. The ActRII signaling inhibitor is administered in an amount sufficient to increase red blood cell count, increase hemoglobin levels, decrease RBC transfusions, promote transfusion independence, treat the cytopenia, improve spleen volume, and / or improve patient-reported outcomes.

[0458] 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, spleen size, and / or patient- reported outcomes (e.g., using MF-SAF-TSS). A finding that the patient’s red blood cell count or hemoglobin levels are increased, that the patient’s spleen size is reduced, or that the patient-reported outcomes 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.

[0459] Other Embodiments

[0460] 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 invention that 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, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

CLAIMS1 . A method of treating a subject having myelofibrosis, the method comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

2. A method of treating a subject with myelofibrosis that has been identified as having a cytopenia, the method comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

3. A method of treating a cytopenia in a subject diagnosed as having myelofibrosis, the method comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

4. A method of treating a subject having polycythemia vera, the method comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

5. The method of claim 1 or 4, wherein the myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination after the subject has been identified as having a cytopenia.

6. The method of claim 1 or 4, wherein the myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination before the subject develops a cytopenia.

7. A method of treating a cytopenia in a subject diagnosed as having polycythemia vera, the method comprising administering in combination to the subject effective amounts of a myelofibrosis therapy and an ActRII signaling inhibitor.

8. A method of treating a subject receiving treatment with a myelofibrosis therapy, the method comprising administering in combination to the subject effective amounts of the myelofibrosis therapy and an ActRII signaling inhibitor.

9. The method of claim 8, wherein the subject has myelofibrosis.

10. The method of claim 8, wherein the subject has polycythemia vera.11 . The method of any one of claims 1 -3, 8, and 9, wherein the subject has intermediate- or high-risk myelofibrosis.

12. The method of any one of claims 1 -3, 8, 9, and 11 , wherein the subject has primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (post-ET MF), or post-polycythemia vera myelofibrosis (post-PV MF).

13. The method of any one of claims 1 -12, wherein the myelofibrosis therapy is not a cytopenia- associated myelofibrosis treatment.

14. The method of any one of claims 1 , 4-6, and 8-10, wherein the subject has a cytopenia.

15. The method of any one of claims 1 , 4-6, 8-10, and 14, wherein the subject is identified as having a cytopenia prior to the administering of the ActRII signaling inhibitor.

16. The method of any one of claims 1 , 4-6, 8-10, 14, and 15, wherein the method further comprises identifying the subject as having a cytopenia prior to the administering of the ActRII signaling inhibitor.

17. The method of any one of claims 2, 3, 7, and 14-16, wherein the cytopenia is anemia.

18. The method of any one of claims 2, 3, 7, and 14-17, wherein the cytopenia is thrombocytopenia.

19. The method of any one of claims 2, 3, 7, and 14-18, wherein the cytopenia is neutropenia.

20. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is a chemotherapeutic agent selected from the group consisting of hydroxyurea, busulfan, selinexor, and cytarabine.21 . The method of any one of claims 1 -19, wherein the myelofibrosis therapy is a kinase inhibitor selected from the group consisting of momelotinib, lestaurtinib, AZD1480, gandotinib, NS-018, BMS- 91 1543, TQ05105, jaktinib, itacitinib, flonoltinib maleate, alisertib, 9-ING-41 , zilurgisertib, everolimus, rineterkib, parsaclisib, buparlisib, umbralisib, roginolisib, TL-895, fostamatinib, rigosertib, dasatinib, TP- 3654, PIM447, and ribociclib.

22. The method of claim 21 , wherein the kinase inhibitor is momelotinib.

23. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is an immunotherapy selected from the group consisting of thalidomide, lenalidomide, pomalidomide, interferon alfa, peginterferon alfa-2b, ropeginterferon alfa-2b, elotuzumab, and canakinumab.

24. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is a hypomethylating agent selected from the group consisting of azacitidine and decitabine.

25. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is a histone deacetylase inhibitor selected from the group consisting of givinostat and panobinostat.

26. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is a bromodomain and extra-terminal protein inhibitor selected from the group consisting of pelabresib, BMS-986158, mivebresib, and ABBV-744.

27. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is a Hedgehog inhibitor selected from the group consisting of glasdegib, I PI-926, sonidegib, and vismodegib.

28. The method of any one of claims 1 -19, wherein the myelofibrosis therapy is zoledronic acid, danazol, LCL-161 , navtemadlin, crizanlizumab, siremadlin, sabatolimab, NIS793, bomedemstat, DISC-0974, zinpentraxin alfa, AVID200, tagraxofusp, pevonedistat, plitidepsin, GB2064, PXS-5505, tipifarnib, reparixin, or enasidenib.

29. The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is an activin A antibody or an antigen binding fragment thereof.

30. The method of claim 29, wherein the activin A antibody is garetosmab.31 . The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is a myostatin antibody or an antigen binding fragment thereof.

32. The method of claim 31 , wherein the myostatin antibody is domagrozumab, landogrozumab, trevogrumab, or apitegromab.

33. The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is an ActRII antibody or an antigen binding fragment thereof.

34. The method of claim 33, wherein the ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861 .

35. The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is an ActRII ligand trap.

36. The method of claim 35, wherein the ActRII ligand trap is an ActRIIA ligand trap.

37. The method of claim 36, wherein the ActRIIA ligand trap is a composition of Table 21 .

38. The method of claim 36, wherein the ActRIIA ligand trap is sotatercept.

39. The method of claim 35, wherein the ActRII ligand trap is an ActRIIB ligand trap.

40. The method of claim 39, wherein the ActRIIB ligand trap is BIIB110, ALG-802, luspatercept, ramatercept, or ACE-2494.41 . The method of claim 39, wherein the ActRIIB ligand trap is a composition of Table 22.

42. The method of claim 35, wherein the ActRII ligand trap is an ActRII chimera ligand trap.

43. The method of claim 42, wherein the ActRII chimera ligand trap is a composition of Table 23 or Table24.

44. The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is an activin B antibody or an antigen binding fragment thereof.

45. The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is a GDF-11 antibody or an antigen binding fragment thereof.

46. The method of any one of claims 1 -28, wherein the ActRII signaling inhibitor is an anti-myostatin adnectin recombinant protein.

47. The method of claim 46, wherein the anti-myostatin adnectin recombinant protein is taldefgrobep alfa.

48. The method of claim 47, wherein the anti-myostatin adnectin recombinant protein comprises a sequence having at least 90% sequence identity to a sequence provided in Table 20.

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