Methods of Using Activin Receptor Type II Signaling Inhibitors - Patent application

Abstract

The present invention features a method for treating a subject undergoing cytopenia-associated myelofibrosis treatment by co-administering an activin receptor type II (ActRII) signaling inhibitor. The ActRII signaling inhibitor can be an antibody that binds to an ActRII ligand, an ActRII antibody, or an ActRII ligand trap.

Description

[Technical field]

[0001] The present invention relates to methods of using activin receptor type II signaling inhibitors. [Background technology]

[0002] Myelofibrosis is a chronic myeloproliferative malignancy characterized by clonal proliferation of bone marrow cells and megakaryocytic hyperplasia / dysplasia resulting in bone marrow fibrosis and osteosclerosis. It may present as a de novo disorder (primary myelofibrosis, PMF) or evolve from polycythemia vera (post-PV MF), essential thrombocythemia (post-ET MF), myelodysplastic syndromes (MDS), lupus, or other hematological and solid tumors. Myeloproliferative neoplasms arise from a single somatically mutated hematopoietic stem cell precursor that clonally expands and gives rise to virtually all myeloid cells as well as B cells and natural killer cells. It is characterized by myelofibrosis, ineffective hematopoiesis, splenomegaly, extramedullary hematopoiesis, systemic symptoms, and shortened survival. While there is no curative medical therapy for patients with myelofibrosis, JAK inhibitors such as ruxolitinib (JAKAFI® / JAKAVI®), fedratinib (INREBIC®), and pacritinib (VONJO™) have been shown to reduce spleen volume and ameliorate symptoms associated with MF. However, JAK inhibitors interfere with normal hematopoiesis, and treatment with ruxolitinib and fedratinib can be complicated by the development of anemia and thrombocytopenia, leading to dose reductions and poor adherence, thereby limiting the number of patients who can remain on JAK inhibitors. Summary of the Invention [Problem to be solved by the invention]

[0003] Thus, there is a need for new therapeutic approaches to prevent or reduce the onset of cytopenias in subjects treated with JAK inhibitors. [Means for solving the problem]

[0004] The present invention provides a method for co-administering an activin receptor type II (ActRII) signaling inhibitor with a cytopenia-associated myelofibrosis treatment, which can be used to treat myelofibrosis, polycythemia vera, or steroid-refractory graft-versus-host disease, or treat cytopenia in subjects with any of these conditions. These methods can be used to reduce adverse reactions associated with treatment with a cytopenia-associated myelofibrosis treatment, improve treatment adherence, improve treatment duration, maintain dose intensity, or increase the dose of a cytopenia-associated myelofibrosis treatment, or reduce cytopenic episodes, transfusion burden, bleeding events, infections, and treatment interruptions or discontinuations of a cytopenia-associated myelofibrosis treatment. The ActRII signaling inhibitor can be an antibody that binds to an ActRII ligand, an anti-ActRII antibody, or an ActRII ligand trap, and exemplary cytopenia-associated myelofibrosis treatments include ruxolitinib (JAKAFI® / JAKAVI®), fedratinib (INREBIC®), pacritinib (VONJO™), and imetelstat.

[0005] Exemplary embodiments of the present invention are described in the paragraphs listed below. E1. A method of treating a subject having myelofibrosis, comprising administering to the subject an effective amount of a cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor.

[0006] E2. The method of E1, wherein the cytopenia associated myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination after the subject is identified as having cytopenia.

[0007] E3. The method of E1, wherein the cytopenia associated myelofibrosis treatment and the ActRII signaling inhibitor are administered in combination before the subject develops cytopenia (e.g., to prevent or reduce the onset of cytopenia).

[0008] E4. A method of treating a subject with myelofibrosis identified as having cytopenia, comprising administering to the subject an effective amount of a cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0009] E5. A method of treating cytopenia in a subject diagnosed with myelofibrosis, comprising administering to the subject an effective amount of a cytopenia associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0010] E6. A method of treating a subject with polycythemia vera (e.g., an adult subject who has had an inadequate response to or is intolerant to hydroxyurea), comprising administering to the subject an effective amount of a cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0011] E7. A method of treating a subject having steroid-refractory graft-versus-host disease (e.g., acute graft-versus-host disease), comprising administering to the subject an effective amount of a cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor.

[0012] E8. The method of E6 or E7, wherein the cytopenia associated myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination after the subject is identified as having cytopenia.

[0013] E9. The method of E6 or E7, wherein the cytopenia associated myelofibrosis therapy and the ActRII signaling inhibitor are administered in combination before the subject develops cytopenias (e.g., to prevent or reduce the onset of cytopenias).

[0014] E10. A method of treating cytopenias in a subject diagnosed with polycythemia vera, comprising administering to the subject an effective amount of a cytopenia associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0015] E11. A method for treating cytopenia in a subject diagnosed with steroid-refractory graft-versus-host disease, comprising administering to the subject an effective amount of a cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0016] E12. A method of treating a subject undergoing treatment with a cytopenia-associated myelofibrosis therapy, comprising administering to the subject an effective amount of a cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0017] E13. A method for improving adherence to treatment with a cytopenia-associated myelofibrosis treatment in a subject in need of a cytopenia-associated myelofibrosis treatment, comprising administering the cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor.

[0018] E14. A method for increasing the dose of a cytopenia-associated myelofibrosis therapy administered to a subject in need of such therapy, comprising administering the cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor (e.g., the subject can receive a higher dose when the two agents are administered simultaneously than when the cytopenia-associated myelofibrosis therapy is administered alone).

[0019] E15. A method for extending the duration of a cytopenia-associated myelofibrosis treatment in a subject in need thereof, comprising administering the cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor (e.g., the subject can continue to receive the cytopenia-associated myelofibrosis treatment for a longer period of time when the two agents are administered simultaneously than when the cytopenia-associated myelofibrosis treatment is administered alone).

[0020] E16. A method for maintaining dose intensity of a cytopenia-associated myelofibrosis therapy in a subject in need of a cytopenia-associated myelofibrosis therapy, comprising administering the cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor (e.g., the subject does not need to reduce the dose of the cytopenia-associated myelofibrosis therapy when the two agents are administered simultaneously, or requires a smaller or lesser dose reduction than when the cytopenia-associated myelofibrosis therapy is administered alone).

[0021] E17. A method for reducing episodes of cytopenia associated with a cytopenia-associated myelofibrosis treatment in a subject in need of a cytopenia-associated myelofibrosis treatment, comprising administering the cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor.

[0022] E18. A method for reducing transfusion burden in a subject treated with a cytopenia-associated myelofibrosis therapy, comprising administering said cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0023] E19. A method for reducing bleeding events in a subject treated with a cytopenia-associated myelofibrosis therapy, comprising administering said cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0024] E20. A method of reducing infection in a subject treated with a cytopenia-associated myelofibrosis therapy, comprising administering the cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0025] E21. A method for reducing treatment interruptions or discontinuations of a cytopenia-associated myelofibrosis treatment in a subject in need thereof, comprising administering the cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor.

[0026] E22. A method for resuming treatment with a cytopenia-associated myelofibrosis treatment in a subject who has developed a myelofibrosis treatment-associated cytopenia (e.g., after cessation of treatment), comprising administering the cytopenia-associated myelofibrosis treatment in combination with an ActRII signaling inhibitor.

[0027] E23. A method for promoting transfusion independence in a subject treated with a cytopenia-associated myelofibrosis therapy, comprising administering said cytopenia-associated myelofibrosis therapy in combination with an ActRII signaling inhibitor.

[0028] E24. The method according to any one of E1 to E5 and E12 to E23, wherein the subject has medium-risk or high-risk myelofibrosis. E25. The method of any one of E1-E5 and E12-E24, wherein the subject has primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (post-ET MF), or post-polycythemia vera myelofibrosis (post-PV MF).

[0029] E26. The method according to any one of E6, E8 to E10, and E12 to E24, wherein the subject has polycythemia vera. E27. The method according to any one of E7 to E9 and E11 to E24, wherein said subject has steroid-refractory graft-versus-host disease.

[0030] E28. The method of any one of E1-E27, wherein said cytopenia associated myelofibrosis treatment is a JAK inhibitor or imetelstat. E29. The method of E28, wherein said JAK inhibitor is ruxolitinib, fedratinib, or pacritinib.

[0031] E30. The method of any one of E1, E6, E7, and E12-E29, wherein the subject has cytopenia. E31. The method of any one of E1, E6, E7, and E12-E30, wherein the subject is identified as having cytopenia prior to administration of the ActRII signaling inhibitor.

[0032] E32. A method according to any one of E1, E6, E7, and E12 to E30, wherein the method further comprises identifying the subject as having cytopenia prior to administration of the ActRII signaling inhibitor.

[0033] E33. The method according to any one of E2 to E5, E8 to E11, E17, and E29 to E32, wherein said cytopenia is anemia. E34. The method according to any one of E2 to E5, E8 to E11, E17, and E29 to E33, wherein said cytopenia is thrombocytopenia.

[0034] E35. The method according to any one of E2 to E5, E8 to E11, E17, and E29 to E34, wherein said cytopenia is neutropenia. E36. The method of any one of E1 to E35, wherein the ActRII signaling inhibitor is an activin A antibody or an antigen-binding fragment thereof.

[0035] E37. The method of E36, wherein said activin A antibody is garetosumab. E38. The method of E36, wherein the activin A antibody or 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 the HCVR sequence of 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 the LCVR sequence of Table 1 (e.g., a HCVR sequence of Table 1 and a LCVR sequence of Table 1, such as a HCVR sequence and a LCVR sequence in the same row of Table 1).

[0036] E39. The method of E36 or E38, wherein the activin A antibody or antigen-binding fragment thereof has a light chain CDR1, CDR2, and CDR3, and a heavy chain CDR1, CDR2, and CDR3, as listed in Table 2 (e.g., the light chain CDR1, CDR2, and CDR3 sequences, and the heavy chain CDR1, CDR2, and CDR3 sequences in the same row of Table 2).

[0037] E40. The method of any one of E1 to E35, wherein the ActRII signaling inhibitor is a myostatin antibody or an antigen-binding fragment thereof. E41. The method of E40, wherein said myostatin antibody is domagrozumab, landgrozumab, trevoglumab, or SRK-015.

[0038] E42. The method of E40, wherein the myostatin antibody or 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 the HCVR sequence of Table 3 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 the LCVR sequence of Table 3 (e.g., a HCVR sequence of Table 3 and a LCVR sequence of Table 3, such as a HCVR sequence and a LCVR sequence in the same row of Table 3, or a HCVR sequence of any one of SEQ ID NOs: 448 to 476 and a LCVR sequence of any one of SEQ ID NOs: 477 to 486).

[0039] E43. The method of E40 or E42, wherein the myostatin antibody or antigen-binding fragment thereof has light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3, as listed in Table 4, Table 5, or Table 6 (e.g., the light chain CDR1, CDR2, and CDR3 sequences, and the heavy chain CDR1, CDR2, and CDR3 sequences in the same row of Table 4).

[0040] E44. The method of any one of E40, E42, and E43, wherein the myostatin antibody or antigen-binding fragment thereof has heavy chain and light chain sequences having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) to the heavy chain and light chain sequences provided in Table 7 (e.g., the heavy chain and light chain sequences in the same row of Table 7).

[0041] E45. The method of any one of E1 to E35, wherein the ActRII signaling inhibitor is an ActRII antibody or an antigen-binding fragment thereof. E46. The method of E45, wherein said ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861.

[0042] E47. The method of E45, wherein the ActRII antibody or 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 the HCVR sequence in Table 8 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 the LCVR sequence in Table 8 (e.g., a HCVR sequence in Table 8 and a LCVR sequence in Table 8, such as a HCVR sequence and a LCVR sequence in the same row of Table 8).

[0043] E48. The method of E45 or E47, wherein the ActRII antibody or antigen-binding fragment thereof has light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3, as listed in Table 9 (e.g., the light chain CDR1, CDR2, and CDR3 sequences, and the heavy chain CDR1, CDR2, and CDR3 sequences in the same row of Table 9).

[0044] E49. The method of any one of E45, E47, and E49, wherein the ActRII antibody or antigen-binding fragment thereof has heavy chain and light chain sequences having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) to the heavy chain and light chain sequences provided in Table 10 (e.g., the heavy chain and light chain sequences in the same row of Table 10).

[0045] E50. The method of any one of E1 to E35, wherein the ActRII signaling inhibitor is an ActRII ligand trap. E51. The method of E50, wherein said ActRII ligand trap is an ActRIIA ligand trap.

[0046] E52. The method of E51, wherein the ActRIIA ligand trap is a composition of Table 18 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 18). E53. The method of E51, wherein the ActRIIA ligand trap comprises the extracellular portion of wild-type ActRIIA (e.g., sequence number 73 or sequence number 729).

[0047] E54. The method of E51, wherein said ActRIIA ligand trap is sotatercept. E55. The method of E50, wherein said ActRII ligand trap is an ActRIIB ligand trap.

[0048] E56. The method of E55, wherein the ActRIIB ligand trap comprises the extracellular portion of wild-type ActRIIB (e.g., SEQ ID NO: 74 or a portion thereof). E57. The method of E55, wherein said ActRIIB ligand trap is BIIB110, ALG-802, luspatercept, ramatercept, or ACE-2494.

[0049] E58. The method of E55, wherein the ActRIIB ligand trap is a composition of Table 19 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 19). E59. The method described in E55, wherein the ActRIIB ligand trap comprises any one of SEQ ID NOs: 745 to 750 (e.g., any one of SEQ ID NOs: 745 to 750 fused via a linker to a portion such as an Fc domain or an Fc domain monomer).

[0050] E60. The method of E50, wherein said ActRII ligand trap is an ActRII chimeric ligand trap. E61. The method of E60, wherein said ActRII ligand trap is a composition of Table 20 or Table 21 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 20 or Table 21).

[0051] E62. The method of any one of E1 to E35, wherein the ActRII signaling inhibitor is an activin B antibody or an antigen-binding fragment thereof. E63. The method of E62, wherein the activin B antibody or 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.

[0052] E64. The method of any one of E1 to E35, wherein said ActRII signaling inhibitor is a GDF-11 antibody or an antigen-binding fragment thereof. E65. The method of any one of E1-E64, wherein the method further comprises evaluating a red blood cell or platelet parameter after administration of the ActRII signaling inhibitor.

[0053] E66. The method of any one of E1-E65, wherein the method results in an increase in hemoglobin of 1.5 g / dL or more (e.g., an increase in hemoglobin of 1.5 g / dL or more 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 the ActRII signaling inhibitor compared to baseline or pre-treatment measurements).

[0054] E67. The method of any one of E1-E66, wherein the method results in a reduction in transfusion burden during the treatment period (e.g., a reduction in RBC units transfused during 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 of treatment with an ActRII signaling inhibitor compared to an 8 week baseline prior to treatment).

[0055] E68. The method of any one of E1-E67, wherein the subject achieves at least 12 weeks of transfusion independence during treatment (e.g., compared to pre-treatment transfusion data for the 12 weeks immediately preceding treatment).

[0056] E69. The ActRII signaling inhibitor increases red blood cell levels, increases hemoglobin levels, increases red blood cell production, increases red blood cell counts, increases hematocrit, reduces transfusion burden, promotes transfusion independence, increases mean corpuscular volume, increases mean corpuscular hemoglobin, increases reticulocyte hemoglobin, increases erythropoietin levels, increases thrombopoietin levels, increases maturation and / or differentiation of erythroid progenitors (e.g., early and / or late erythroid progenitors), increases late erythroid precursor maturation, mobilizes early progenitors to the erythroid lineage, increases reticulocytes, increases proerythroblast counts, decreases accumulation of erythroid progenitors, increases early erythroid precursor and / or progenitor cell counts, promotes progression of erythroid precursors and / or progenitors through erythropoiesis, treats anemia, or increases platelet levels. the method of any one of E1-E68, wherein the compound is administered in an amount sufficient to increase platelet volume, increase the immature platelet fraction, increase platelet precursors, increase platelet production, increase platelet count, increase or induce megakaryocyte differentiation and / or maturation, increase megakaryocyte precursor cell regeneration, decrease platelet precursor cell accumulation, improve blood clotting, decrease bleeding events, reduce intradermal bleeding, treat thrombocytopenia, increase neutrophil levels, increase neutrophil production, increase neutrophil count, increase or induce differentiation and / or maturation of precursor cells into neutrophils, treat neutropenia, decrease infection susceptibility, affect myostatin, activin A, activin B, and / or BMP9 signaling in the subject, or decrease or inhibit binding of activin A, activin B, and / or myostatin to their receptors (e.g., their endogenous receptors).

[0057] E70. The method of any one of E1 to E5 and E12 to E69, wherein the ActRII signaling inhibitor is administered in an amount sufficient to reduce spleen volume, reduce bone marrow fibrosis, reduce osteosclerosis, improve bone marrow fibrosis grade, or reduce high platelet levels.

[0058] E71. The method of any one of E1-E70, wherein said method does not cause a vascular complication in said subject. E72. The method of E71, wherein said method does not increase vascular permeability or leakage.

[0059] E73. The method of any one of E1-E72, wherein the subject is a human. definition To facilitate understanding of the present invention, certain terms are defined below. Terms defined herein have meanings commonly understood by those skilled in the art to which the present invention pertains. Terms such as "a", "an", and "the" are not intended to refer to a singular entity only, but are intended to include a general class within which a specific example may be used for illustration. Although the terms herein are used to describe certain embodiments of the present invention, their use does not limit the present invention, except as outlined in the claims.

[0060] As used herein, the term "about" refers to a value within 10% above or below the stated value. As used herein, any value provided in a range of values ​​includes both the upper and lower limits, as well as any value subsumed within those limits.

[0061] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., an ActRII signaling inhibitor as described herein) to a subject by any effective route. Exemplary administration routes are described herein below.

[0062] The term "antibody" is used in the broadest sense and specifically includes 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.

[0063] An "antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding or variable region of an 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.

[0064] As used herein, the term "extracellular activin receptor type IIA (ActRIIA) variant" refers to a peptide comprising a soluble extracellular portion of ActRIIA, a single transmembrane receptor, having at least one amino acid substitution compared to wild-type extracellular ActRIIA (e.g., the bolded portion of the sequence of SEQ ID NO: 75 shown below). The sequence of wild-type human ActRIIA precursor protein is shown below (SEQ ID NO: 75), where the signal peptide is in italics and the extracellular portion is in bold.

[0065] Wild-type human ActRIIA precursor protein (SEQ ID NO:75):

[0066] [ka]

[0067] The extracellular ActRIIA variant may have the sequence of any one of SEQ ID NOs: 1-72. In certain embodiments, the extracellular ActRIIA variant has the sequence of any one of SEQ ID NOs: 6-72 (Table 12). In some embodiments, the extracellular ActRIIA variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73).

[0068] As used herein, the term "cytopenia associated myelofibrosis therapy" refers to a drug that is either approved for the treatment of myelofibrosis or in clinical development for the treatment of myelofibrosis and is associated with the development of cytopenias (e.g., anemia, thrombocytopenia, or neutropenia) as an adverse reaction.

[0069] As used herein, the term "linker" refers to a link between two elements, e.g., between peptides or protein domains. The ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to a moiety (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72). The moiety may increase the stability or improve the pharmacokinetic properties of the polypeptide. The moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide via a linker. The linker may 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 type of bond created by 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 sequence of 1-200 amino acids) that occurs between two elements, e.g., between 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 a peptide spaced apart through the polypeptide backbone). For example, the formation of a disulfide bond between two hinge regions that form an Fc domain is not considered a linker.

[0070] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain comprises at least C H 2 domain and C H The Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) with a human Fc domain that includes a three-domain antibody constant domain. The Fc domain monomer comprises a second antibody constant domain and a third antibody constant domain (C H 2 and C H3). In some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not include any portion of an immunoglobulin that can act as an antigen recognition region, e.g., a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are composed of two CDRs. H The dimerization occurs through interactions between the three antibody constant domains, as well as one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack effector functions, typical of a "dead Fc domain." In certain embodiments, the Fc domain monomers within the Fc domain are each mutated to reduce the interaction or binding between the Fc domain and the Fcγ receptor. H In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0071] As used herein, the term "albumin-binding peptide" refers to an amino acid sequence of 12-16 amino acids that has affinity for serum albumin and plays a role in binding to serum albumin. Albumin-binding peptides can be of different origins, e.g., human, mouse, or rat. In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 83).

[0072] As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein or fragment thereof of the extracellular matrix that binds to transmembrane receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID number P02751 (SEQ ID NO: 82). In other embodiments, the fibronectin domain is an adnectin protein.

[0073] As used herein, the term "human serum albumin" refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood. It accounts for approximately half of the serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID number P02768 (SEQ ID NO:81).

[0074] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., humans) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cell, e.g., a human red blood cell, platelet, neutrophil, or muscle cell).

[0075] As used herein, the term "fused" is used to describe the combination or joining 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 can be fused to form one continuous protein structure, e.g., a polypeptide, by chemical conjugation, chemical bonds, peptide linkers, or any other covalent bonding means. In some embodiments of the ActRII ligand trap described herein, the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras 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)) is linked via a linker to a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), an Fc domain (e.g., the sequence of SEQ ID NO: 84 or SEQ ID NO: 79), 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 the N-terminus or C-terminus of human serum albumin (e.g., the sequence of SEQ ID NO: 81). For example, the extracellular ActRIIA variant may be fused to a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a peptide linker, where the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIA variant via 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., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a chemical bond, e.g., a peptide bond.

[0076] 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 a sequence of any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70). 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 positions of wild-type ActRIIA. Exemplary C-terminal extensions are the amino acid sequence NP (2 amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 78) (6 amino acid C-terminal extension). Any amino acid sequence that does not interfere with the activity of the polypeptide can be used. SEQ ID NO: 71, which is the sequence of SEQ ID NO: 69 with the C-terminal extension of NP, and SEQ ID NO: 72, which is the sequence of SEQ ID NO: 69 with the C-terminal extension of NPVTPK (SEQ ID NO: 78), represent two possible ways in which the polypeptides of the invention can be modified to include a C-terminal extension.

[0077] 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 as necessary to achieve the maximum percent identity (i.e., gaps can be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for purposes of determining percent identity can be achieved in a variety of ways within the skill of the art, for example, 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 required to achieve maximum alignment over the entire 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 be translated as a given candidate sequence having or containing a certain percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows: 100×(Fraction A / B) where A is the number of amino acid (or nucleic acid) residues scored as identical in an alignment of the candidate and reference sequences, and 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 is 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 is not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0078] In certain embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits 50% to 100% identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes is, for example, 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. If 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.

[0079] As used herein, the term "serum half-life" refers to the time required for the plasma concentration of a therapeutic protein in a subject to be reduced by half in the context of administration to the subject.Proteins can be redistributed or removed from the bloodstream, or can be degraded, for example, by proteolysis.Comparing serum half-life can be done by comparing the serum half-life of Fc fusion proteins.

[0080] As used herein, the term "affinity" or "binding affinity" refers to the strength of binding interaction between two molecules. In general, binding affinity refers to the strength of the total non-covalent interactions between a molecule and its binding partner, e.g., an extracellular ActRIIA variant and BMP9 or activin A. Unless otherwise indicated, binding affinity refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally determined by the dissociation constant (K D ) or affinity constant (K A ) Two molecules that have low binding affinity for each other generally bind slowly and tend to dissociate easily, with a large K D Two molecules that have a high affinity for each other generally bind more easily and tend to remain bound longer, exhibiting a small K D The K of the two interacting molecules D can be determined using methods and techniques well known in the art, for example, surface plasmon resonance.D is k off / k on It is calculated as a ratio of

[0081] As used herein, the phrase "affecting myostatin, activin A, activin B, and / or BMP9 signaling" refers to altering the binding of myostatin, activin A, activin B, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII (e.g., ActRIIA, e.g., endogenous ActRIIA). In some embodiments, a polypeptide comprising an extracellular ActRIIA variant described herein reduces or inhibits the binding of myostatin, activin A, activin B, and / or BMP9 to their receptors, e.g., ActRIIA, ActRIIB, and / or BMPRII (e.g., ActRIIA, e.g., endogenous ActRIIA).

[0082] As used herein, the terms "increase" and "decrease" refer to the regulation of the amount of a function, expression, or activity of a metric, respectively, greater or less, compared to a reference. For example, after administration of a polypeptide of the present invention comprising an extracellular ActRIIA variant in the methods described herein, the amount of a marker of a metric described herein (e.g., hemoglobin level, red blood cell count, hematocrit, reticulocyte count, platelet count, or transfusion burden) may increase or decrease in a subject compared to the amount of the marker before administration. In general, the metric is measured after administration when the administration produces the listed effect, for example, at least one week, one month, three months, or six months after starting a treatment regimen.

[0083] As used herein, the terms "increasing red blood cell levels" and "promoting erythropoiesis" refer to clinically observable metrics such as hematocrit, red blood cell count, and hemoglobin measurements, and are intended to be neutral with respect to the mechanism by which such changes occur. The terms "erythropoiesis" and "erythropoiesis" refer to the production of red blood cells, such as the process of erythropoiesis in which red blood cells are produced in the bone marrow.

[0084] As used herein, the term "anemia" refers to any abnormality in hemoglobin or red blood cells that results in a decrease in oxygen levels in blood. Anemia may be related to abnormal production, processing, or performance of red blood cells and / or hemoglobin. The term anemia refers to any decrease in the number of red blood cells and / or the level of hemoglobin in blood compared to normal blood levels. For example, a subject who has a hemoglobin level of 10 g / dL or less or who has received a red blood cell (RBC) transfusion can be identified as having anemia.

[0085] As used herein, the terms "increasing platelet levels" and "promoting platelet formation" refer to a clinically observable metric, such as platelet count, and are intended to be neutral with respect to the mechanism by which such a change occurs. The terms "platelet formation" and "platelet production" refer to the production of platelets, such as the process by which platelets are produced from megakaryocytes.

[0086] As used herein, the terms "increasing neutrophil levels" and "promoting neutrophil formation" refer to clinically observable metrics such as neutrophil counts and are intended to be neutral with respect 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 by which neutrophils are produced in the bone marrow.

[0087] As used herein, the term "thrombocytopenia" refers to a condition in which blood contains fewer than normal numbers of platelets, which may result from failure to produce platelets, accumulation of platelets in an enlarged spleen, or destruction of platelets. Normal platelet levels range from about 150,000 to 450,000 per microliter of blood in humans. A platelet count below 150,000 per microliter is lower than normal. If the platelet count falls below 50,000 per microliter of blood, bleeding may occur after relatively minor injury, and if the platelet count falls below 10,000 to 20,000 per microliter of blood, severe bleeding may occur without any recognized injury.

[0088] 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 neutrophil count is approximately 1500 cells per microliter of blood. Below this level, there is an increased risk of infection. The severity of neutropenia is classified as mild (1000-1500 neutrophils per microliter of blood), moderate (500-1000 neutrophils per microliter of blood), and severe (less than 500 neutrophils per microliter of blood). There are many causes of neutropenia, but it is typically classified into two major categories: destruction or depletion of neutrophils faster than the bone marrow can produce new neutrophils, or a decrease in the production of neutrophils in the bone marrow.

[0089] As used herein, the term "ineffective hematopoiesis" refers to a failure to produce fully mature hematopoietic cells (e.g., failure to produce red blood cells, platelets, and neutrophils). Ineffective hematopoiesis can result from single or multiple defects, such as abnormal proliferation and / or differentiation of progenitor cells (e.g., excessive production of progenitor cells that cannot complete differentiation), which can lead to hyperproliferation or insufficiency of progenitor cells.

[0090] As used herein, the terms "erythropoiesis stimulating agents" and "ESAs" refer to a class of drugs that act on the proliferative phase of erythroid development by expanding the pool of early progenitor cells. Examples of erythropoiesis stimulating agents are epoetin alfa and darbepoetin alfa.

[0091] As used herein, the term "vascular complications" refers to any damage to blood vessels, such as vascular disorders or damage to the blood vessel wall. Damage to the blood vessel wall can cause increased vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the blood vessel wall to allow the flow of small molecules, proteins, and cells into and out of the blood vessel. Increased vascular permeability or leakage can be caused by an increase in gaps between endothelial cells lining the blood vessel wall (e.g., an increase in the size and / or number of gaps) and / or a thinning of the blood vessel wall.

[0092] As used herein, the term "polypeptide" refers to a single polymer in which the monomers are amino acid residues covalently conjugated to each other through amide bonds. Polypeptides are intended to encompass any amino acid sequence, whether naturally occurring, recombinant, or synthetically produced.

[0093] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. These two identical monomers may form homodimers by covalent or non-covalent bonds. For example, an Fc domain may be a homodimer of two Fc domain monomers when the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein that includes an extracellular ActRIIA variant fused to an Fc domain monomer may form a homodimer by the interaction of the two Fc domain monomers that form the Fc domain within the homodimer.

[0094] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. These two monomers can form a heterodimer by covalent or non-covalent bonds. For example, a polypeptide described herein that comprises an extracellular ActRIIA variant fused to an Fc domain monomer can form a heterodimer by the interaction of two Fc domain monomers, each fused to a different ActRIIA variant, that form an Fc domain within the heterodimer.

[0095] As used herein, the term "host cell" refers to a vehicle that contains the necessary cellular components, e.g., organelles, required for expressing a protein from a corresponding nucleic acid. The nucleic acid is typically contained in a nucleic acid vector that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell can 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).

[0096] As used herein, the term "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid, or vector of the invention, or an amount of a pharmaceutical composition comprising a polypeptide, nucleic acid, or vector of the invention, effective to achieve a desired therapeutic effect in treating a patient with a disease or condition, such as myelofibrosis or a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis treatment. Specifically, a therapeutically effective amount of a polypeptide, nucleic acid, or vector avoids adverse side effects.

[0097] As used herein, the term "pharmaceutical composition" refers to a medicinal or pharmaceutical preparation that includes an active ingredient, as well as excipients and diluents that can make the active ingredient suitable for the method of administration. The pharmaceutical composition of the present invention includes pharma- ceutical acceptable ingredients that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition can be in the form of a tablet or capsule for oral administration, or in aqueous form for intravenous or subcutaneous administration.

[0098] As used herein, the term "pharmaceutical acceptable carrier or excipient" refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutical acceptable carrier must be compatible with other ingredients of the formulation and not harmful to the recipient. In the present invention, a pharmaceutical acceptable carrier or excipient must provide sufficient pharmaceutical stability for the polypeptides described herein (e.g., ActRII signaling inhibitors, such as ActRII ligand traps, including extracellular ActRIIA variants), nucleic acid molecule(s) encoding the polypeptide, or vectors containing such nucleic acid molecule(s). The nature of the carrier or excipient will vary depending on the method of administration. For example, for intravenous administration, aqueous carriers are generally used, and for oral administration, solid carriers are preferred.

[0099] As used herein, the term "treating and / or preventing" refers to the treatment and / or prevention of a disease or condition associated with myelofibrosis or myelofibrosis therapy, such as cytopenia (e.g., anemia, thrombocytopenia, or neutropenia), using the methods and compositions of the present invention. Generally, the treatment of a disease or condition associated with myelofibrosis or myelofibrosis therapy, such as cytopenia (e.g., anemia, thrombocytopenia, or neutropenia), is performed after the subject has developed the disease or condition. The prevention of a disease or condition associated with myelofibrosis or myelofibrosis therapy, such as cytopenia (e.g., anemia, thrombocytopenia, or neutropenia), refers to a step or procedure performed when the subject is at risk of developing the disease or condition. A subject may exhibit signs or mild symptoms that are determined by a physician to be an indication of or a risk factor for developing the disease or condition, have another disease or condition that is associated with the development of the disease or condition, are undergoing a treatment that may cause the disease or condition, or have a family history or genetic predisposition to developing the disease or condition but have not yet developed the disease or condition.

[0100] As used herein, the term "subject" refers to a mammal, such as, preferably, a human. Mammals include, but are not limited to, humans, as well as domestic and livestock animals, such as monkeys (e.g., cynomolgus monkeys), mice, dogs, cats, horses, and cows. [Brief description of the drawings]

[0101] [Figure 1] 1 is a series of graphs showing the effect of ActRIIA / B-mFc (SEQ ID NO: 69 fused to a mouse Fc domain via a linker) on platelet production. As shown in FIG. 1, ActRIIA / B-mFc increased circulating platelet counts and bone marrow megakaryocyte progenitor cells within 12 hours after administration. Data are presented as mean±standard error of the mean. Statistical analysis was performed using Student's T-test. *p<0.05, ****p<0.0001. [Diagram 2]A series of graphs showing that treatment with ActRIIA / B-mFc directly affected megakaryocyte maturation. Treatment of 11-week-old C57 / Bl6 mice with ActRIIA / B-mFc (10 mg / kg subcutaneously) increased the number of CD41+ megakaryocyte progenitors at 12 hours post-treatment (left) and polyploid megakaryocytes at 24 hours post-treatment (right). Data are presented as the mean ± standard error of the mean. [Figure 3A] FIG. 3 is a series of graphs showing that treatment with ActRIIA / B-mFc accelerated recovery of platelet counts after platelet depletion compared to vehicle treatment. Eleven-week-old mice were treated with either anti-GP1bα (0.08 mg / kg, Efferet) or IgG control. Four days after treatment, the anti-GP1bα-treated group was further divided to receive either vehicle or ActRIIA / B-mFc (7.5 mg / kg) treatment. Platelets were measured at the indicated time points after anti-GP1bα dosing. Ten days after treatment, mice were euthanized and bone marrow cells were harvested. As shown in FIG. 3A, mice treated with ActRIIA / B-mFc exhibited accelerated recovery of platelet counts after platelet depletion compared to vehicle-treated mice in this immune thrombocytopenic mouse model. In addition, as shown in Figures 3B-3C, the number of CD41+ megakaryocyte progenitors in bone marrow of the ActRIIA / B-mFc-treated group was increased by 25% compared to the vehicle-treated group, with higher 4N ploidy levels at day 10 after platelet depletion. For platelet data, statistical analysis was performed using repeated measures mixed effects modeling. Individual comparisons shown are from Tukey post-hoc tests. For CD41 data, statistical analysis was performed using one-way ANOVA and individual comparisons calculated using Tukey post-hoc tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3B]FIG. 3 is a series of graphs showing that treatment with ActRIIA / B-mFc accelerated recovery of platelet counts after platelet depletion compared to vehicle treatment. Eleven-week-old mice were treated with either anti-GP1bα (0.08 mg / kg, Efferet) or IgG control. Four days after treatment, the anti-GP1bα-treated group was further divided to receive either vehicle or ActRIIA / B-mFc (7.5 mg / kg) treatment. Platelets were measured at the indicated time points after anti-GP1bα dosing. Ten days after treatment, mice were euthanized and bone marrow cells were harvested. As shown in FIG. 3A, mice treated with ActRIIA / B-mFc exhibited accelerated recovery of platelet counts after platelet depletion compared to vehicle-treated mice in this immune thrombocytopenic mouse model. In addition, as shown in Figures 3B-3C, the number of CD41+ megakaryocyte progenitors in bone marrow of the ActRIIA / B-mFc-treated group was increased by 25% compared to the vehicle-treated group, with higher 4N ploidy levels at day 10 after platelet depletion. For platelet data, statistical analysis was performed using repeated measures mixed effects modeling. Individual comparisons shown are from Tukey post-hoc tests. For CD41 data, statistical analysis was performed using one-way ANOVA and individual comparisons calculated using Tukey post-hoc tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3C]FIG. 3 is a series of graphs showing that treatment with ActRIIA / B-mFc accelerated recovery of platelet counts after platelet depletion compared to vehicle treatment. Eleven-week-old mice were treated with either anti-GP1bα (0.08 mg / kg, Efferet) or IgG control. Four days after treatment, the anti-GP1bα-treated group was further divided to receive either vehicle or ActRIIA / B-mFc (7.5 mg / kg) treatment. Platelets were measured at the indicated time points after anti-GP1bα dosing. Ten days after treatment, mice were euthanized and bone marrow cells were harvested. As shown in FIG. 3A, mice treated with ActRIIA / B-mFc exhibited accelerated recovery of platelet counts after platelet depletion compared to vehicle-treated mice in this immune thrombocytopenic mouse model. In addition, as shown in Figures 3B-3C, the number of CD41+ megakaryocyte progenitors in bone marrow of the ActRIIA / B-mFc-treated group was increased by 25% compared to the vehicle-treated group, with higher 4N ploidy levels at day 10 after platelet depletion. For platelet data, statistical analysis was performed using repeated measures mixed effects modeling. Individual comparisons shown are from Tukey post-hoc tests. For CD41 data, statistical analysis was performed using one-way ANOVA and individual comparisons calculated using Tukey post-hoc tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4] FIG. 1 is a series of graphs showing that treatment with a single dose of ActRIIA / B-mFc resulted in an increase in circulating platelets for at least 85 days. 11-week-old C57BL / 6 mice were treated with a single dose of either vehicle or ActRIIA / B-mFc (10 mg / kg) via subcutaneous administration. Separate cohorts of mice from both dosing groups were sampled for whole blood on study days 37, 51, and 85, and platelet counts were determined using a veterinary hematology analyzer (Heska Element HT5). Data are presented as mean ± standard error of the mean. Statistical analysis was performed using Student's T-test. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Diagram 5]Figure 1 shows that ex vivo treatment with ActRIIA / B-mFc reversed activin-mediated changes in megakaryocyte progenitor cells. Bone marrow cells from 11-week-old C57Bl / 6 mice were isolated and treated with activin A (5 mg / kg), ActRIIA / B-mFc (10 mg / kg), or a combination of both for 6 days. Cells were harvested after 6 days and analyzed using flow cytometry (N=2). Error bars = standard error of the mean. [Figure 6] Graph showing that treatment with anti-activin A antibody increased platelets in wild type mice. Ten-week-old C57Bl / 6 male mice were administered either TBS (vehicle), anti-activin A (5 mg / kg), or ActRIIA / B-mFc (10 mg / kg) intraperitoneally. Whole blood was sampled 24 hours after administration and platelet counts were determined using a veterinary hematology analyzer (Hematrue). Data are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. **p<0.01, ***p<0.001. [Figure 7] A series of graphs showing that platelet count and platelet volume were increased in a TPO-high model of myelofibrosis, and treatment with ActRIIA / B-mFc attenuated platelet proliferation. Seven-week-old C57Bl / 6 albino mice (B6(Cg)-Tyr, Jackson Laboratory) were tail vein injected with 0.75 mg / kg of thrombopoietin (TPO) expression plasmid cloned into pLEV113 plasmid (Lake Pharma). The injection was performed using a hydrodynamic approach with a 100 mL / kg volume injected over a short period of time (6-10 seconds). On day 3 after TPO injection, mice were divided into two groups and administered either vehicle (TBS) or ActRIIA / B-mFc (7.5 mg / kg) by IP injection twice weekly. On day 14 after TPO injection, mice were sacrificed. Hematological parameters were analyzed by measuring using a Heska Element HT5 veterinary hematology analyzer. N=10-12 mice / group. Results are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. ****=p<0.0001. [Figure 8] FIG. 11 is a series of graphs showing that ActRIIA / B-mFc ameliorated TPO-induced anemia. The TPO-high model of myelofibrosis became anemic 14 days after TPO expression and exhibited reduced red blood cell, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentrations. Treatment with ActRIIA / B-mFc was associated with significant improvements in RBC metrics and appeared to reduce the development of anemia in this model. N=10-12 mice / group. Results are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 9] Graph showing that ActRIIA / B-mFc reduced TPO-induced splenic extramedullary hematopoiesis. In the high TPO model of myelofibrosis, the bone marrow is less able to produce hematopoiesis due to the expansion of megakaryocyte growth and proliferation, inducing compensatory extramedullary hematopoiesis in the liver and spleen. These data show a significant reduction in splenomegaly in mice treated with ActRIIA / B-mFc, indicating a reduction in splenic extramedullary hematopoiesis, likely due to a reduced need for this compensatory process. N=10-12 mice / group. Results are presented as mean±standard error of the mean. Statistical analysis was performed using one-way ANOVA. *=p<0.05, ****=p<0.0001. [Figure 10] FIG. 1 is a series of graphs showing that ActRIIA / B-mFc reduced TPO-mediated increases in leukocytes and lymphocytes. The TPO-high model of myelofibrosis exhibited increases in leukocytes, neutrophils, and lymphocytes. Treatment with ActRIIA / B-mFc reduced the increases in leukocytes and lymphocytes in TPO-high mice. N=10-12 mice / group. Results are presented as mean±standard error of the mean. Statistical analysis was performed using one-way ANOVA. ***=p<0.001, ****=p<0.0001. [Figure 11]Figure 1 shows that ActRIIA / B-mFc increased body weight in ruxolitinib-treated mice. Female C57Bl / 6 mice aged 10-12 weeks were orally administered either vehicle (n=10) or ruxolitinib at 90 mg / kg (n=20) or 120 mg / kg (n=20) twice daily. After 37 days of ruxolitinib therapy, blood was sampled by buccal bleeding for hematological evaluation. On day 41, mice from each ruxolitinib dosing were split into two groups to receive either TBS (vehicle) (n=10) or ActRIIA / B-mFc (7.5 mg / kg, n=10 / group) by IP twice weekly for 14 days, concurrent with the continuation of ruxolitinib dosing. Hematological parameters were assessed in whole blood using a Heska Element HT5 veterinary hematology analyzer. N=10. Data are presented as mean ± standard error of the mean. Two-way ANOVA was used for statistical analysis. **=p<0.01 between vehicle and ruxolitinib 90 mg / kg + ActRIIA / B-mFc at each time point. ***=p<0.005 between vehicle and ruxolitinib 90 mg / kg + ActRIIA / B-mFc at each time point. ****=p<0.0001 between vehicle and ruxolitinib 90 mg / kg + ActRIIA / B-mFc at each time point. #=p<0.05 between vehicle and ruxolitinib 120 mg / kg + ActRIIA / B-mFc at each time point. ##=p<0.01 between vehicle and ruxolitinib 120 mg / kg + ActRIIA / B-mFc at each time point. [Figure 12]

[0023] Figure 1 is a series of graphs showing that administration of ruxolitinib resulted in a decrease in red blood cell volume, hemoglobin, and hematocrit. Data are presented as the mean ± standard error of the mean. Vehicle N=10, ruxolitinib 90 N=20, ruxolitinib 120 N=20. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc test. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 13]

[0023] Figure 1 is a series of graphs showing that ActRIIA / B-mFc reversed ruxolitinib-associated decreases in red blood cell volume, hemoglobin, and hematocrit. Data are presented as mean ± standard error of the mean. N=10 for all groups. Statistical analysis was performed using one-way ANOVA followed by Tukey post-hoc test. ns=not significant, *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 14A] 14 is a series of graphs showing expression of TGF-β receptors and ligands in megakaryocyte progenitor cells from naive mice. Mouse bone marrow megakaryocyte progenitor cells expressed activin, GDF, BMP, and TGF-β ligands (FIG. 14B), as well as their cognate receptors (FIG. 14A). Receptors and ligands directly related to ActRIIA / B-mFc are bolded. ND=not detectable. [Figure 14B] 14 is a series of graphs showing expression of TGF-β receptors and ligands in megakaryocyte progenitor cells from naive mice. Mouse bone marrow megakaryocyte progenitor cells expressed activin, GDF, BMP, and TGF-β ligands (FIG. 14B), as well as their cognate receptors (FIG. 14A). Receptors and ligands directly related to ActRIIA / B-mFc are bolded. ND=not detectable. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] The present invention features a method of co-administering an activin receptor type II (ActRII) signaling inhibitor with a cytopenia-associated myelofibrosis treatment. The ActRII signaling inhibitor can be an antibody that binds to an ActRII ligand, an anti-ActRII antibody, or an ActRII ligand trap, and exemplary cytopenia-associated myelofibrosis treatments include ruxolitinib (JAKAFI® / JAKAVI®), fedratinib (INREBIC®), pacritinib (VONJO™), and imetelstat. These methods can be used to treat myelofibrosis, such as intermediate-risk 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 may also reduce or ameliorate cytopenias (e.g., anemia, thrombocytopenia, and / or neutropenia) resulting from cytopenia-associated myelofibrosis treatment, thereby improving adherence to, duration of, and dose intensity of, cytopenia-associated myelofibrosis treatment, reducing treatment interruptions or discontinuations, and reducing episodes of cytopenia associated with cytopenia-associated myelofibrosis treatment.

[0103] ActRII Signaling Activin type II receptor is a single transmembrane domain receptor that regulates the signal of the ligand of transforming growth factor beta (TGF-β) superfamily. The ligand of TGF-β superfamily is involved in many physiological processes, such as muscle growth, blood vessel growth, cell differentiation, homeostasis, and bone formation. Examples of the ligand of TGF-β superfamily include, for example, activin A, activin B, inhibin, growth differentiation factor (GDF) (e.g., GDF8, also known as myostatin, and GDF11), and bone morphogenetic protein (BMP) (e.g., BMP9).

[0104] The TGF-β signaling pathway regulates hematopoiesis, the activin-involved signaling pathway prevents the differentiation of erythrocyte, platelet, and neutrophil progenitors, maintaining progenitor cells in a quiescent state, and the BMP-involved signaling pathway promotes the differentiation of progenitor cells. Homeostasis of this process is essential to ensure that all cell types, including erythrocytes, leukocytes, and platelets, are adequately replenished in the blood. Relatedly, the activin receptor ligand GDF11 has been found to be overexpressed in a mouse model of hemolytic anemia and associated with defective 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 the activin receptor itself, may disrupt hematopoiesis. Thus, methods of reducing or inhibiting activin A, activin B, and / or myostatin signaling can be used to promote hematopoiesis and treat diseases and conditions associated with ineffective hematopoiesis, such as cytopenias (e.g., anemia, thrombocytopenia, or neutropenia) associated with myelofibrosis.

[0105] The present invention relates, in part, to an ActRIIA ligand trap, including an ActRIIA variant, that increases platelet and megakaryocyte progenitor cells, promotes megakaryocyte maturation, ameliorates TPO-induced anemia, reduces TPO-induced extramedullary hematopoiesis in the spleen, and inhibits TPO-induced hematopoiesis in myelofibrosis. 高Based on the inventors' findings that the TPO-mediated increase in white blood cells and lymphocytes in the model was reduced, and the ruxolitinib-associated decrease in RBC volume, hematocrit, and hemoglobin was reversed. The inventors also found that the inhibition of activin A by anti-activin A antibody increases platelets. These data suggest that the co-administration of an ActRII signaling inhibitor and a thrombocytopenia-associated myelofibrosis treatment can improve or ameliorate the thrombocytopenia associated with the thrombocytopenia-associated myelofibrosis treatment, which can address the adverse reactions associated with the thrombocytopenia-associated myelofibrosis treatment, improve the adherence to the treatment, treatment duration, and dose intensity of the thrombocytopenia-associated myelofibrosis treatment, and reduce the treatment interruption or discontinuation and episodes of thrombocytopenia associated with the thrombocytopenia-associated myelofibrosis treatment.

[0106] ActRII signaling inhibitors An ActRII signaling inhibitor is an agent that reduces or prevents the interaction of an ActRII ligand with ActRIIA and / or ActRIIB by binding to either the ligand or the receptor. ActRII signaling inhibitors for use in the methods described herein are provided herein below.

[0107] 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 galetusumab (also known as REGN-2477). Additional activin A antibodies that can be used in the methods described herein include those described in International Patent Publication Nos. WO2015017576, WO2013074557, and WO2008031061, US Patent Application No. US2015 / 0359850, 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.

[0108] In some embodiments, the activin A antibody or antigen-binding fragment thereof has a heavy chain variable region (HCVR) and a light chain variable region (LCVR) listed in Table 1 (e.g., the HCVR and LCVR in the same row of Table 1). In some embodiments, the activin A antibody or antigen-binding fragment thereof comprises an HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the HCVR sequences in Table 1, e.g., SEQ ID NOs: 138, 140, 142, 143, 144, 146, 148, 150, 151, 172, and 174, and an LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the LCVR sequences in Table 1, e.g., SEQ ID NOs: 139, 141, 145, 147, 149, 173, and 175. In some embodiments, the activin A antibody or antigen-binding fragment thereof has HCVR and LCVR sequences that have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) with the HCVR and LCVR sequences listed in Table 1, excluding the light chain CDR1, CDR2, and CDR3, and the heavy chain CDR1, CDR2, and CDR3. In some embodiments, the activin A antibody or antigen-binding fragment thereof has the light chain CDR1, CDR2, and CDR3, and the heavy chain CDR1, CDR2, and CDR3 sequences of the HCVR and LCVR sequences of Table 1. In some embodiments, the activin A antibody or antigen-binding fragment thereof comprises the HCVR and LCVR sequences of the same row of Table 1.

[0109] [Table 1-1]

[0110] [Table 1-2]

[0111] In some embodiments, the activin A antibody or antigen-binding fragment thereof has the CDR sequences set forth in Table 2 (i.e., light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3). In some embodiments, the activin A antibody or antigen-binding fragment thereof has 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 any one of the light chain variable CDR1 sequences of Table 2, e.g., any one of SEQ ID NOs: 155, 161, 179, and 185, any one of the light chain variable CDR2 sequences of Table 2, e.g., any one of SEQ ID NOs: 156, 162, 180, and 186. 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 any one of the light chain variable CDR3 sequences in Table 2, e.g., SEQ ID NOs: 157, 163, 181, and 187; 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 any one of the heavy chain variable CDR1 sequences of Table 2, e.g., any one of SEQ ID NOs: 152, 158, 176, and 182; 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 any one of the heavy chain variable CDR2 sequences of Table 2, e.g., any one of SEQ ID NOs: 153, 159, 177, and 183; and 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 any one of the heavy chain variable CDR3 sequences of Table 2, e.g., SEQ ID NOs: 154, 160, 178, and 184.In some embodiments, the activin A antibody or antigen-binding fragment thereof comprises the light chain CDR1, CDR2, and CDR3 sequences, and the heavy chain CDR1, CDR2, and CDR3 sequences of the same row of Table 2.

[0112] [Table 2]

[0113] In some embodiments, the ActRII signaling inhibitor is a myostatin antibody or an antigen-binding fragment thereof. In some embodiments, the myostatin antibody is domaglozumab (also known as PF-06252616), landlozumab (also known as LY2495655), trevoglumab (also known as REGN-1033), or SRK-015. Additional myostatin antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2007047112, WO2007044411, WO2006116269, WO2012024242, WO2016073853, WO2013024 ... Nos. 186719, WO2009058346, WO2011150008, WO2016168613, WO2007024535, and WO2016098357, U.S. Patent Application Nos. US20070178095 and US20210246198, and U.S. Pat. No. 0, No. 10,738,111, No. 7,632,499, No. 8,066,995, No. 7,635,760, No. 7,745,583, No. 7, No. 745,583, No. 7,807,159, No. 8,999,343, No. 10,307,480, No. 8,992,913, No. 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.

[0114] In some embodiments, the myostatin antibody or antigen-binding fragment thereof has a HCVR and LCVR listed in Table 3 (e.g., the HCVR and LCVR in the same row of Table 3). In some embodiments, the myostatin antibody or antigen-binding fragment thereof has a HCVR sequence in Table 3, e.g., SEQ ID NOs: 164, 188, 201, 204-210, 222-228, 234, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 298, 306, 308, 310, 312, 314, 316, 318, 320, 356, 371, 373, 387, 389, 391, 405, 407, 409, 411, 413, 415, 417, 419, 421-423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 444, 446, and 448-476 and at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of any one of HCVR sequences having sequence identity and LCVR sequences in Table 3, e.g., 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, and 477-486. In some embodiments, the myostatin antibody or antigen-binding fragment thereof has HCVR and LCVR sequences that have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to the HCVR and LCVR sequences listed in Table 3, excluding light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3.In some embodiments, the myostatin antibody or antigen-binding fragment thereof has light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3 sequences of the HCVR and LCVR sequences of Table 3. In some embodiments, the myostatin antibody or antigen-binding fragment thereof comprises the HCVR sequence and the LCVR sequence of the same row of Table 3. In some embodiments, the myostatin antibody or antigen-binding fragment thereof comprises the HCVR sequence of any one of SEQ ID NOs: 448-476 and the LCVR sequence of any one of SEQ ID NOs: 477-486.

[0115] [Table 3-1]

[0116] [Table 3-2]

[0117] [Table 3-3]

[0118] [Table 3-4]

[0119] [Table 3-5]

[0120] [Table 3-6]

[0121] [Table 3-7]

[0122] [Table 3-8]

[0123]

Table 3-9

[0124]

Table 3-10

[0125]

Table 3-11

[0126]

Table 3-12

[0127]

Table 3-13

[0128]

Table 3-14

[0129]

Table 3-15

[0130] In some embodiments, the myostatin antibody or antigen-binding fragment thereof has a CDR sequence set forth in Table 4, Table 5, or Table 6 (i.e., light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3). In some embodiments, the myostatin antibody or antigen-binding fragment thereof has a CDR sequence that is at least 90% identical (e.g., at least 90% identical) to any one of the light chain variable CDR1 sequences in Table 4 or Table 6, e.g., SEQ ID NOs: 169, 193, 198, 238, 241, 303, 325, 330, 362, 378, 384, 396, 402, 826, 490, 493, and 343-346. light chain variable CDR1 sequences having a sequence identity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to a light chain variable CDR2 sequence in Table 4 or Table 6, such as SEQ ID NOs: 170, 194, 199, 239, 304, 326, 331, 363, 379, 385, 397, 403, 827, 491, and 347 to 34 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 any one of SEQ ID NOs: 171, 195, 200, 240, 245, 249, 305, 327, 364, 380, 386, 398, 404, 828, 492, and 350 to 355; 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 any one of SEQ ID NOs: 166, 190, 171, 195, 200, 240, 245, 249, 305, 327, 364, 380, 386, 398, 404, 828, 492, and 350 to 355;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 any one of SEQ ID NOs: 196, 235, 242, 246, 300, 322, 328, 359, 366, 375, 381, 393, 399, 823, 487, and 332-334, a heavy chain variable CDR2 sequence of Table 4 or Table 5, e.g., any one of SEQ ID NOs: 167, 191, 197, 236, 243, 247, 301, 323, 329, 360, 365, 376, 382, ​​394, 400, 824, 488, 335, and 336 and 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 CDR3 sequence of Table 4 or Table 5, e.g., any one of SEQ ID NOs: 168, 192, 237, 244, 248, 302, 324, 361, 377, 383, 395, 401, 825, 489, and 337-342. In some embodiments, the myostatin antibody or antigen-binding fragment thereof comprises the light chain CDR1, CDR2, and CDR3 sequences, and the heavy chain CDR1, CDR2, and CDR3 sequences of the same row of Table 4.

[0131] [Table 4-1]

[0132] [Table 4-2]

[0133] [Table 4-3]

[0134] [Table 4-4]

[0135] [Table 5]

[0136] [Table 6]

[0137] In some embodiments, the myostatin antibody or antigen-binding fragment thereof has heavy and light chain sequences that have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) to the heavy and light chain sequences provided in Table 7. In some embodiments, the myostatin antibody or antigen-binding fragment thereof has heavy and light chain sequences in the same row of Table 7. In some embodiments, the heavy and light chains are selected from the group consisting of SEQ ID NOs:274 and 275, SEQ ID NOs:276 and 277, SEQ ID NOs:278 and 279, SEQ ID NOs:280 and 281, SEQ ID NOs:282 and 283, SEQ ID NOs:284 and 285, SEQ ID NOs:286 and 287, SEQ ID NOs:288 and 289, SEQ ID NOs:290 and 291, SEQ ID NOs:292 and 293, SEQ ID NOs:294 and 295, SEQ ID NOs:296 and 297, SEQ ID NOs:367 and 368, or SEQ ID NOs:369 and 370. and the light chain has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the sequence of the first SEQ ID NO in each pair, and the light chain has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the sequence of the second SEQ ID NO in each pair.

[0138] [Table 7-1]

[0139]

Table 7-2

[0140]

Table 7-3

[0141]

Table 7-4

[0142]

Table 7-5

[0143]

Table 7-6

[0144]

Table 7-7

[0145] In some embodiments, the myostatin antibody is a bispecific antibody that also binds activin A. Exemplary bispecific myostatin antibodies that may be used in the methods described herein include those described in U.S. 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 bispecific antibody binds to activin A in Table 1. HCVR and LCVR (e.g., HCVR sequences having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the HCVR sequences in Table 1, e.g., SEQ ID NOs: 138, 140, 142, 143, 144, 146, 148, 150, 151, 172, and 174, and LCVR sequences in Table 1, e.g., SEQ ID NOs: 139, 141, 142, 143, 144, 146, 148, 150, 151, 172, and 174, 45, 147, 149, 173, and 175) and myostatin HCVRs and LCVRs in Table 3 (e.g., HCVR sequences in Table 3, e.g., 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 to 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 444, 446, and 448 to 476 and HCVR sequences having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to one of the LCVR sequences in Table 3, e.g., 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, and 477 to 486). In some embodiments, the bispecific antibody comprises an activin A heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 of Table 2 (e.g., an activin A heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 of the same row in Table 2), and a myostatin heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 of Table 4 (e.g., a myostatin heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 of the same row in Table 4). In some embodiments, the bispecific antibody comprises an activin A HCVR (SEQ ID NO: 138) and LCVR (SEQ ID NO: 139), and a myostatin HCVR (SEQ ID NO: 164) and LCVR (SEQ ID NO: 165). In some embodiments, the bispecific antibody comprises activin A HCVR (SEQ ID NO: 138) and LCVR (SEQ ID NO: 139), and myostatin HCVR (SEQ ID NO: 387) and LCVR (SEQ ID NO: 388). In some embodiments, the bispecific antibody comprises activin A HCVR (SEQ ID NO: 138) and LCVR (SEQ ID NO: 139), and myostatin HCVR (SEQ ID NO: 391) and LCVR (SEQ ID NO: 392). In some embodiments, the bispecific antibody comprises activin A HCVR (SEQ ID NO: 144) and LCVR (SEQ ID NO: 145), and myostatin HCVR (SEQ ID NO: 164) and LCVR (SEQ ID NO: 165). In some embodiments, the bispecific antibody comprises activin A HCVR (SEQ ID NO: 144) and LCVR (SEQ ID NO: 145), and myostatin HCVR (SEQ ID NO: 387) and LCVR (SEQ ID NO: 388). In some embodiments, the bispecific antibody comprisesActivin A HCVR (SEQ ID NO: 144) and LCVR (SEQ ID NO: 145), and myostatin HCVR (SEQ ID NO: 391) and LCVR (SEQ ID NO: 392). In some embodiments, the bispecific antibody comprises Activin A heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 (SEQ ID NOs: 152-157), and myostatin heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 (SEQ ID NOs: 166-171). In some embodiments, the bispecific antibody comprises activin A heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 (SEQ ID NOs: 158-163), and myostatin heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 (SEQ ID NOs: 166-171).

[0146] In some embodiments, the ActRII signaling inhibitor is an activin B antibody or an antigen-binding fragment thereof. Activin B antibodies that can be used in the methods described herein include those described in U.S. 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 that includes three CDRs from the HCVR sequence of SEQ ID NO: 494 and a LCVR that includes 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 that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO: 494. In some embodiments, the activin B antibody or 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.

[0147] MKCSWIMFFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMYWVKQRPGQGLEWIGMIHPNSGSTNYNGKFKTGATLTVDKSSSTVYMQLSSLTSEDSAVYYCARWGYGGNYDYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSL (sequence number 494) MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISTMEAEDAVTYYCHQYHRSPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK (SEQ ID NO: 495) In some embodiments, the ActRII signaling inhibitor is a GDF-11 antibody or an antigen-binding fragment thereof.

[0148] In some embodiments, the ActRII signaling inhibitor is an ActRII antibody or an antigen-binding fragment thereof. There are 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 Publication Nos. WO2010125003, WO2012064771, WO2017156488, WO2013063536, WO2018175460, WO2021044287, WO2013188448, WO2020243448, U.S. Patent Application Nos. US20180066061, US20180230221, US20180111991, and US20200181271, each of which is incorporated herein by reference. , US20210309749, and US20160200818, as well as U.S. Pat. Nos. 9,453,080, 10,266,598, 10,981,999, 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.

[0149] In some embodiments, the ActRII antibody or antigen-binding fragment thereof has a HCVR and LCVR listed in Table 8 (e.g., the HCVR and LCVR in the same row of Table 8). In some embodiments, the ActRII antibody or antigen-binding fragment thereof has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10% identity with any one of the HCVR sequences in Table 8, e.g., 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. 0%) sequence identity to any one of the LCVR sequences in Table 8, and LCVR sequences that have at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the LCVR sequences in Table 8, such as 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 antigen-binding fragment thereof has HCVR and LCVR sequences that have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to the HCVR and LCVR sequences listed in Table 8, excluding the light chain CDR1, CDR2, and CDR3, and the heavy chain CDR1, CDR2, and CDR3. In some embodiments, the ActRII antibody or antigen-binding fragment thereof has the light chain CDR1, CDR2, and CDR3, and the heavy chain CDR1, CDR2, and CDR3 sequences of the HCVR and LCVR sequences in Table 8. In some embodiments, the ActRII antibody or antigen-binding fragment thereof comprises the HCVR and LCVR sequences in the same row of Table 8.

[0150] [Table 8-1]

[0151]

Table 8-2

[0152]

Table 8-3

[0153]

Table 8-4

[0154]

Table 8-5

[0155]

Table 8-6

[0156] In some embodiments, the ActRII antibody or antigen-binding fragment thereof has the CDR sequences set forth in Table 9 (i.e., light chain CDR1, CDR2, and CDR3, and heavy chain CDR1, CDR2, and CDR3). In some embodiments, the ActRII antibody or antigen-binding fragment thereof has a light chain variable CDR1 sequence that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the light chain variable CDR1 sequences in Table 9, e.g., SEQ ID NOs: 499, 505, 543, 580, 588, 619, 625, 633, 640, 648, 654, 663, 684, 702, 705, 711, 714, 720, and 726. a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of the light chain variable CDR2 sequences of Table 9, e.g., 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 of Table 9, e.g., SEQ ID NOs: 501, 507, 54 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 any one of SEQ ID NOs: 496, 502, 540, 577, 585, 616, 622, and 728; , 629, 630, 638, 644, 651, 659, 660, 669 to 672, 679 to 681, 699, 708, 717, and 723; 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 any one of SEQ ID NOs: 497, 503, 541, 546, 550 to 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 CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of the heavy chain variable CDR3 sequences of Table 9, e.g., sequences In some embodiments, the ActRII antibody or antigen-binding fragment thereof comprises 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 any one of numbers 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 comprises a light chain CDR1, CDR2, and CDR3 sequence, and a heavy chain CDR1, CDR2, and CDR3 sequence in the same row of Table 9.

[0157] [Table 9-1]

[0158] [Table 9-2]

[0159] [Table 9-3]

[0160] [Table 9-4]

[0161] [Table 9-5]

[0162]

Table 9-6

[0163]

Table 9-7

[0164]

Table 9-8

[0165]

Table 9-9

[0166]

Table 9-10

[0167]

Table 9-11

[0168]

Table 9-12

[0169]

Table 9-13

[0170]

Table 9-14

[0171]

Table 9-15

[0172] In some embodiments, the ActRII antibody or antigen-binding fragment thereof has heavy and light chain sequences that have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) to the heavy and light chain sequences provided in Table 10. In some embodiments, the ActRII antibody or antigen-binding fragment thereof has heavy and light chain sequences in the same row of Table 10. In some embodiments, the heavy and light chains have the sequences of SEQ ID NOs:508 and 509, SEQ ID NOs:510 and 511, SEQ ID NOs:557 and 558, SEQ ID NOs:559 and 560, SEQ ID NOs:561 and 562, SEQ ID NOs:563 and 564, SEQ ID NOs:565 and 566, SEQ ID NOs:567 and 568, SEQ ID NOs:569 and 570, SEQ ID NOs:571 and 572, SEQ ID NOs:573 and 574, or SEQ ID NOs:575 and 576 (e.g., heavy chain the light chain has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the sequence of the first SEQ ID NO in each pair, and the light chain has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the sequence of the second SEQ ID NO in each pair.

[0173] [Table 10-1]

[0174] [Table 10-2]

[0175] [Table 10-3]

[0176] [Table 10-4]

[0177] [Table 10-5]

[0178] [Table 10-6]

[0179] In some embodiments, the ActRII signaling inhibitor is an ActRII ligand trap. An ActRII ligand trap is a polypeptide that includes an extracellular portion of ActRIIA and / or ActRIIB that can bind 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., Fc domain, Fc domain monomer, albumin-binding peptide, fibronectin domain, or human serum albumin) via a linker. An ActRII ligand trap can reduce or inhibit the binding of an ActRII ligand to endogenous activin type II receptor, thereby reducing ActRII signaling. Because ActRII ligand traps include the extracellular portion of the receptor, they are soluble and can bind to and capture ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways.

[0180] In some embodiments, the ActRII ligand trap is an ActRIIA ligand trap.The ActRIIA ligand trap can comprise the extracellular portion of wild-type ActRIIA (e.g., human or mouse ActRIIA), or can comprise the extracellular portion of wild-type ActRIIA that comprises one or more amino acid substitutions compared with wild-type human extracellular ActRIIA.The wild-type amino acid sequence of the extracellular portion of human ActRIIA is shown below.

[0181] Human ActRIIA, extracellular portion (SEQ ID NO:73): GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS The ActRIIA ligand trap may comprise a sequence of SEQ ID NO: 73 or a variant thereof that comprises one or more amino acid substitutions. In some embodiments, the ActRIIA ligand trap comprises a portion of SEQ ID NO: 73 (e.g., a contiguous portion truncated by removal of amino acids from the N-terminus, C-terminus, or both) or a variant thereof that comprises one or more amino acid substitutions. In some embodiments, the ActRIIA ligand trap comprises a sequence of SEQ ID NO: 73 or a portion thereof that has additional amino acids at the C-terminus from the wild-type sequence of ActRIIA (SEQ ID NO: 75). Exemplary sequences of portions of wild-type ActRIIA proteins that are truncated at the N-terminus and include additional amino acids at the C-terminus from SEQ ID NO: 75 that can be included in the ActRIIA ligand trap are provided below.

[0182] ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP (SEQ ID NO: 729) Studies have shown that BMP9 binds to ActRIIB with approximately 300-fold higher binding affinity than ActRIIA (see, for example, Townson et al., J.Biol.Chem.287:27313,2012). ActRIIA-Fc is known to have a longer half-life compared to ActRIIB-Fc. ActRIIA ligand traps, including extracellular ActRIIA variants constructed by introducing amino acid residues of ActRIIB into ActRIIA, are described herein below, with the aim of imparting the physiological properties imparted by ActRIIB while also maintaining the beneficial physiological and pharmacokinetic properties of ActRIIA. The optimal peptide promotes hematopoiesis (e.g., increases red blood cell count, hemoglobin level, hematocrit, reticulocyte, platelet level (e.g., platelet count), and / or neutrophil level (e.g., neutrophil count)) while retaining, for example, low binding affinity to BMP9 and longer serum half-life as an Fc fusion protein. Preferred ActRIIA variants exhibit similar or improved binding to activin and / or myostatin compared to wild-type extracellular ActRIIA, enabling them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling.These variants can be used to increase hemoglobin levels, hematocrit, red blood cell counts (e.g., to increase red blood cell production and / or red blood cell mass or volume), erythroid progenitor maturation and / or differentiation (e.g., maturation and / or differentiation of early or late (e.g., terminal) erythroid progenitors into proerythroblasts, reticulocytes, or erythrocytes), decrease erythroid progenitor accumulation (e.g., by stimulating progenitor cells to mature), increase late precursor (erythroid precursor) maturation (e.g., terminal maturation, e.g., maturation of reticulocytes into erythrocytes, or maturation of erythroblasts into reticulocytes and / or erythrocytes), mobilize early progenitor cells into the erythroid lineage, increase early erythroid precursor and / or progenitor cell numbers, promote erythroid precursor and / or progenitor cell proliferation through erythropoiesis, and / or promote cell proliferation through erythropoiesis. Cytopenias (e.g., anemia, thrombocytopenia, and / or neutropenia) associated with myelofibrosis or myelofibrosis treatment can be treated by promoting cell progression (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 cell renewal, and / or platelet production), increasing megakaryocyte progenitors, decreasing platelet progenitor accumulation (e.g., by stimulating progenitor cells to mature), increasing neutrophil levels (e.g., increasing neutrophil count, e.g., increasing neutrophil production), and / or increasing differentiation and / or maturation of progenitor cells (e.g., myeloid progenitor cells, myeloblasts, or myelocytes) into neutrophils. In some embodiments, amino acid substitutions can be introduced into the extracellular ActRIIA variant to decrease or eliminate the binding affinity of the variant to BMP9.

[0183] The ActRIIA ligand trap described herein can include an extracellular ActRIIA variant having at least one amino acid substitution compared to a wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 73. Possible amino acid substitutions at 27 different positions can be introduced into the extracellular ActRIIA variant (Table 11). In some embodiments, the extracellular ActRIIA variant can have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or more) amino acid sequence identity with the sequence of the wild-type extracellular ActRIIA (SEQ ID NO: 73). The extracellular ActRIIA variant may have one or more (e.g., 1-27, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) amino acid substitutions compared to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 73). In some embodiments, the extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of SEQ ID NO: 1) may include amino acid substitutions at all 27 positions listed in Table 11. In some embodiments, the extracellular ActRIIA variant may contain amino acid substitutions at some, e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 of the 27 positions listed in Table 11.

[0184] Amino acid substitutions can worsen or improve the activity and / or binding affinity of the ActRIIA variants of the present invention. In order to maintain polypeptide function, X in the sequences shown in Tables 11 and 12 (SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) can be used. 17 It is important that the lysine (K) at position 1 is retained. Substitution at that position may result in loss of activity. For example, An ActRIIA variant having GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 85) has reduced activity in vivo and 17 This indicates that substitution of lysine (K) at position X with alanine (A) is not tolerated. Thus, the ActRIIA variants of the present invention, including the variants in Tables 11 and 12 (e.g., SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), are characterized in that the amino acid K is substituted with X. 17 Maintain position.

[0185] The ActRIIA variants of the present invention preferably have reduced or weak BMP9 binding, or substantially no BMP9 binding. 23 Place, X 24 Place, X 25 th, and X 26 is decreased (e.g., decreased compared to wild-type ActRIIA) in an ActRIIA variant comprising the amino acid sequence TEEN (SEQ ID NO: 76) at position X 24 Maintain amino acid K at position X 23 Place, X 24 Place, X 25 th, and X 26 The sequence TEEN (SEQ ID NO: 76) and the sequence TKEN (SEQ ID NO: 77) can be used interchangeably in ActRIIA variants of the invention (e.g., variants of Tables 11 and 12, e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to provide reduced BMP9 binding.

[0186] [Table 11]

[0187] The ActRIIA variants of the present invention may further include a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension may add one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus of any of the variants shown in Tables 11 and 12 (e.g., SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). The C-terminal extension may correspond to a sequence from the same position of wild-type ActRIIA. One possible C-terminal extension that can be included in the ActRIIA variants of the present invention is the amino acid sequence NP. For example, a sequence including the C-terminal extension NP is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). Another exemplary C-terminal extension that can be included in the ActRIIA variants of the present invention is the 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)).

[0188] In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X 3 is E and X 6 is R and X 11 is D and X 12 is K and X 13 is R and X 16 is K or R, and X 17 is K and X 19 is W and X 20 is L and X 21 is D and X 22 is I or F. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1, X 2 is Y and X 4 is L and X 8 is E and X 9 is E and X 14 is L and X 18 is K and X 23 is T and X 25 is E and X 26 is N and X 27is Q. These substitutions in SEQ ID NO: 1 can also be made in SEQ ID NOs: 2-5. In some embodiments of an extracellular ActRIIA variant having the sequence of SEQ ID NO: 1, X 1 is F or Y, and X 2 is Y and X 4 is L and X 5 is D or E, and X 7 is P or R, and X 8 is E and X 9 is E and X 10 is K or Q, and X 14 is L and X 15 is F or Y, and X 16 is K or R, and X 18 is K and X 22 is I or F, and X 23 is T and X 24 is K or E, and X 25 is E and X 26 is N and X 27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1, X 1 is F or Y, and X 2 is Y and X 3 is E and X 4 is L and X 5 is D or E, and X 6 is R and X 7 is P or R, and X 8 is E and X 9 is E and X 10 is K or Q, and X 11 is D and X 12 is K and X 13 is R and X 14 is L and X 15 is F or Y, and X 16 is K or R, and X 17 is K and X 18 is K and X 19 is W and X 20 is L and X 21 is D and X 22 is I or F, and X 23is T and X 24 is K or E, and X 25 is E and X 26 is N and X 27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1 or 2, X 17 is K. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 1-3, X 17 is K and X 23 is T and X 24 is E and X 25 is E and X 26 is N. In some embodiments of the extracellular ActRIIA variant having any one of SEQ ID NOs: 1-5, X 17 is K and X 23 is T and X 24 is K and X 25 is E and X 26 is N.

[0189] In some embodiments, the ActRIIA ligand trap described herein comprises an extracellular ActRIIA variant having any one of SEQ ID NOs: 6-72 (Table 12).

[0190] [Table 12-1]

[0191] [Table 12-2]

[0192] [Table 12-3]

[0193] [Table 12-4]

[0194] [Table 12-5]

[0195] [Table 12-6]

[0196] In some embodiments, the ActRIIA ligand trap comprises an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). 17 It has the amino acid K at position X. 17 Changing the amino acid at position 1444 may result in reduced activity. For example, an ActRIIA variant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 85) has reduced activity in vivo and 17 This indicates that substitution of A for K at position 1 is not permitted.

[0197] In some embodiments, X 23 Place, X 24 Place, X 25 th, and X 26 An ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) having the sequence TEEN (SEQ ID NO: 76) at position X 24 In some embodiments, at position X, the amino acid E can be substituted with the amino acid K. 23 Place, X 24 Place, X 25 th, and X 26 An ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) having the sequence TKEN (SEQ ID NO: 77) at position X 24At position X, the amino acid K may be substituted with the amino acid E. 23 Place, X 24 Place, X 25 th, and X 26 ActRIIA variants having the sequence TEEN (sequence number 76) or TKEN (sequence number 77) at position 1 have reduced or weak BMP9 binding (e.g., reduced BMP9 binding compared to BMP9 binding of wild-type ActRIIA).

[0198] In some embodiments, an ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) may further comprise a C-terminal extension (e.g., one or more additional amino acids at the C-terminus of the ActRIIA variant). The C-terminal extension may correspond to a sequence from the same position of wild-type ActRIIA. In some embodiments, the C-terminal extension is the amino acid sequence NP. For example, a sequence comprising 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 the amino acid sequence NPVTPK (SEQ ID NO: 78). For example, a sequence comprising 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 may add one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus of the ActRIIA variant.

[0199] In some embodiments, an ActRIIA ligand trap comprising an extracellular ActRIIA variant may further comprise a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) that may be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIA variant via a linker or other covalent bond. A polypeptide comprising an extracellular ActRIIA variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or heterodimer) due to interaction between two Fc domain monomers that combine to form the Fc domain in the dimer.

[0200] Additionally, in some embodiments, the ActRIIA ligand traps described herein (e.g., ActRIIA variant-Fc fusion proteins) have a serum half-life of at least 7 days in humans. The ActRIIA ligand traps have a K D In some embodiments, the ActRIIA ligand trap can bind to activin A at X. 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) BMP9 binding (e.g., reduced BMP9 binding compared to BMP9 binding of wild-type ActRIIA). In some embodiments, the ActRIIA ligand trap with reduced or low BMP9 binding has X 23 Place, X 24 Place, X 25 th, and X 26 In some embodiments, the ActRIIA ligand trap has the sequence TEEN (SEQ ID NO: 76) or TKEN (SEQ ID NO: 77) at position 144. In some embodiments, the ActRIIA ligand trap does not substantially bind to human BMP9.

[0201] In some embodiments, the ActRIIA ligand trap has a K of about 800 pM or less. D(e.g., a K 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) D For example, a K of about 800 pM to about 200 pM D In some embodiments, the ActRIIA ligand trap can bind to human activin A with a K of 800 pM or less. D (e.g., a K 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) D For example, a K of about 800 pM to about 200 pM D ) can bind to human activin B. The ActRIIA ligand trap has a K of approximately 5 pM or higher. D (e.g., a K 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 more) D ) and may bind to growth differentiation factor 11 (GDF-11).

[0202] In some embodiments, the ActRIIA ligand trap is sotatercept (also known as ACE-011). Additional ActRIIA ligand traps that can be used in the methods described herein include those described in International Patent Application Publication No. WO2007062188, and U.S. Patent Nos. 7,709,605, 9,138,459, 7,612,041, 8,067,360, 8,629,109, 9,572,865, 9,163,075, 10,071,135, and 7,951,771, each of which is incorporated herein by reference.

[0203] In some embodiments, ActRII ligand trap is ActRIIB ligand trap.ActRIIB ligand trap can comprise the extracellular portion of wild-type ActRIIB (for example, human or mouse ActRIIB), or can comprise the extracellular portion of wild-type ActRIIB that comprises one or more amino acid substitutions compared with wild-type human extracellular ActRIIB.The wild-type amino acid sequence of the extracellular portion of human ActRIIB is shown below.

[0204] Human ActRIIB, extracellular portion (SEQ ID NO:74): GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWL DDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT The ActRIIB ligand trap may include a sequence of SEQ ID NO: 74 or a variant thereof that includes one or more amino acid substitutions. In some embodiments, the ActRIIB ligand trap includes a portion of SEQ ID NO: 74 (e.g., a contiguous portion truncated by removal of amino acids from the N-terminus, C-terminus, or both) or a variant thereof that includes one or more amino acid substitutions. For example, the ActRIIB ligand trap may include a sequence of SEQ ID NO: 74 with a L60D substitution. In another example, the ActRIIB ligand trap may include a sequence of SEQ ID NO: 74 with a substitution at position E9 (e.g., an E9W, E9A, E9F, E9Q, E9V, E9I, E9L, E9M, E9K, E9H, or E9Y substitution), an S25T substitution, and / or an R45A substitution. In some embodiments, the ActRIIB ligand trap is BIIB110 (formerly 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 can be used in the methods described herein include those described in International Patent Application Publication Nos. WO2010 / 062383, WO2015 / 192127, WO2019140283, and WO2021189010, U.S. Patent Application Publication Nos. US20110250198 and US20200407415, and U.S. Pat. Nos. 10,913, 102,513, and 102,513, each of which is incorporated herein by reference. No. 782, No. 8,058,229, No. 8,216,997, No. 8,703,927, No. 9,439,945, No. 9,932,379, No. 10,131,700, No. 10,689,427 No. 10,889,626, No. 10,829,532, No. 10,829,533, No. 8,361,957, No. 9,505,813, No. 10,377,996, No. 9,617,319 , No. 8,710,016, No. 7,709,605, No. 8,252,900, No. 7,842,663, No. 8,343,933, No. 9,399,669, No. 10,259,861, No. 8 , 138,142, 8,178,488, 8,293,881, 9,181,533, 9,745,559, 10,358,633, 11,066,654, 9,61 0,327, 9,284,364, 8,067,562, 8,614,292, 7,947,646, 8,716,459, 8,501,678, 8,999,917, 9,447,165, 9,809,638, 10,407,487, 8,410,043, 9,273,114, and 10,308,704.

[0205] In some embodiments, the ActRIIB ligand trap comprises an ActRIIB variant having the sequence of SEQ ID NO: 730 shown in Table 13.

[0206] [Table 13]

[0207] In some embodiments, the ActRIIB variant has the sequence of any one of SEQ ID NOs: 731-744 (Table 14).

[0208] [Table 14-1]

[0209] [Table 14-2]

[0210] 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). The N-terminal truncation can be made by removing 1-7 amino acids from the N-terminus of an ActRIIB variant shown in Tables 13 and 14. The N-terminal truncation can remove up to two 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 variant). Additional ActRIIB variants with N-terminal truncations are provided below.

[0211] ETRECIYYNANWELERTNQSGLERCYGDKDKRRHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 745) ETRECIYYNANWELERTNQSGLERCEGDQDKRLHCYASWRNSSGTIELVKKGCWLDDINCYDRQECVATKENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 746) ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT (SEQ ID NO: 747) ETRWCIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 748) ETRWCIYYNANWELERTNQTGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 749) ETRYCIYYNANWELERTNQTGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 750) In some embodiments, an ActRIIB ligand trap comprising an ActRIIB variant may further comprise a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) that may be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIB variant via a linker or other covalent bond. An ActRIIB ligand trap comprising an extracellular ActRIIB variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or heterodimer) due to the interaction between the two Fc domain monomers that combine to form the Fc domain in the dimer.

[0212] In some embodiments, the ActRII ligand trap is an ActRII chimeric ligand trap. The ActRII chimeric ligand trap comprises a portion of extracellular ActRIIA (e.g., human ActRIIA) and a portion of extracellular ActRIIB (e.g., human ActRIIB). In some embodiments, the ActRII chimeric ligand trap comprises an N-terminal portion of extracellular ActRIIB (SEQ ID NO: 74 shown above) linked 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 and begins with the next amino acid located at the corresponding position in ActRIIA). In some embodiments, the N-terminus of the ActRII chimera contained in the ActRII chimeric ligand trap comprises the six amino acids found at the N-terminus of extracellular ActRIIA linked to the fifth amino acid of extracellular ActRIIB. In some embodiments, the N-terminus of the ActRII chimera contained in the ActRII chimeric ligand trap begins with the first amino acid located at the N-terminus of the extracellular ActRIIB. In some embodiments, the N-terminus of the ActRII chimera contained in the ActRII chimeric ligand trap includes the first 10 amino acids found at the N-terminus of the extracellular ActRIIA linked to the ninth amino acid of the extracellular ActRIIB. The extracellular ActRII chimera contained in the ActRII chimeric ligand trap can also include one or more amino acid substitutions in the portion of the chimera corresponding to the sequence of ActRIIB compared to the 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 corresponding to the sequence of ActRIIA compared to the wild-type extracellular ActRIIA (e.g., SEQ ID NO: 73 shown above). Amino acid substitutions at nine different positions can be introduced into the extracellular ActRII chimera (Table 15).The extracellular ActRII chimera may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions compared to the sequence of the wild-type sequence (e.g., compared to the sequence of the wild-type extracellular ActRIIB (SEQ ID NO: 74) if a portion of the chimera corresponds to a region of the wild-type extracellular ActRIIB, or compared to the sequence of the wild-type extracellular ActRIIA (SEQ ID NO: 73) if a portion of the chimera corresponds to a region of the wild-type extracellular ActRIIA). Positions where amino acid substitutions can be made, as well as the amino acids that can be substituted at these positions, are listed in Table 15. ActRII chimeric ligand traps that can be used in the methods described herein include those described in International Patent Application Publication No. WO2021189019A1, the disclosure of which is incorporated herein by reference.

[0213] [Table 15-1]

[0214] [Table 15-2]

[0215] In some embodiments, in the ActRII chimeras of SEQ ID NOs: 751-771 (shown in Table 15), X 1 is D and X 2 is I, F, or E, and X 3 is N or T, and X 4 is A or E, and X 5 is T or K, and X 6 is E or K, and X 7 is E or D, and X 8 is N or S, and X 9 is E or Q. In some embodiments, in the extracellular ActRII chimera of SEQ ID NOs: 174-216, X 1 is D and X 2 is I or F, and X 3 is N and X 4 is A or E, and X 5 is T or K, and X6 is E or K, and X 7 is E or D, and X 8 is N or S, and X 9 is E or Q.

[0216] In some embodiments, the ActRII chimeric ligand trap comprises any one of SEQ ID NOs: 772-793 (Table 16).

[0217] [Table 16-1]

[0218] [Table 16-2]

[0219] In some embodiments, the ActRII chimera contained in the ActRII chimeric ligand trap is obtained by replacing one or more amino acid sequences corresponding to β-sheets and optionally one or more intervening sequences (e.g., sequences between β-sheets) from one ActRII protein (e.g., ActRIIB) to the corresponding positions of another ActRII protein (e.g., ActRIIA). For example, an ActRII chimera can be produced by replacing one or more amino acid sequences corresponding to β-sheets in ActRIIB and optionally one or more intervening sequences with amino acid sequences corresponding to β-sheets from ActRIIA and optionally intervening sequences. An ActRII chimera can also be produced by replacing one or more amino acid sequences corresponding to β-sheets in ActRIIA and optionally one or more intervening sequences with amino acid sequences corresponding to β-sheets from ActRIIB and optionally intervening sequences. In an ActRII chimera, a β-sheet and optionally an intervening sequence from one protein is replaced with a corresponding β-sheet and optionally a corresponding intervening sequence from the other protein (e.g., the fifth β-sheet (β 5A ) from ActRIIB, and the fifth β-sheet (β 5BEach ActRII protein has seven β-sheets (β 1 ~β 7 ) and eight intervening sequences (X 1 ~X 8 The ActRII chimera has 1a , β 2a , β 3a , β 4a , β 5a , or β 7a At least one of, and β 1b , β 2b , β 3b , β 4b , β 5b , or β 7b Thus, the ActRII chimera contained in the ActRII chimera ligand trap can have one to five β-sheet substitutions (e.g., one β-sheet substitution from one ActRII protein). 1 , β 2 , β 3 , β 4 , β 5 , and β 7 ActRII chimeras can also have one to seven intervening sequence replacements (e.g., one, two, three, four, or five of the X from one ActRII protein can be replaced with the corresponding β-sheet sequence from the other ActRII protein). 1 , X 2 , X 3 , X 5 , X 6 , X 7 , and X 8 (e.g., one, two, three, four, five, six, or seven of the intervening sequences may be replaced with the corresponding intervening sequences from the other ActRII protein). In some embodiments, the replaced beta-sheet sequence is a minimal beta-sheet sequence (e.g., RHCFATWKNI(β 3a ) (SEQ ID NO: 804) 3b ) (SEQ ID NO: 806) 4a) (SEQ ID NO: 808) 4b ) (SEQ ID NO: 810), at least ELVKKGCW (SEQ ID NO: 811), VEK (β 5a ) at least VE, VAT (β 5b ) at least a part of V, KFSYF(β 7a ) (SEQ ID NO: 819), or RFTHL (β 7b ) (SEQ ID NO: 820) is at least a T that is a portion of the ActRII chimera. The extracellular ActRII chimera is the same length (e.g., has the same number of amino acids) as wild-type extracellular ActRIIA and ActRIIB, so that in embodiments in which the minimal β-sheet sequence is replaced, consecutive amino acids from ActRIIA or ActRIIB are used to connect the minimal β-sheet to adjacent intervening sequences to maintain the length (e.g., number of amino acids) of the ActRII chimera (e.g., so that the extracellular ActRII chimera does not have fewer amino acids than the corresponding regions of extracellular ActRIIA and ActRIIB). Exemplary ActRII chimera sequences that can be included in the ActRII chimeric ligand trap are provided in Table 17. ActRII chimeric ligand traps that can be used in the methods described herein include those described in International Patent Application No. PCT / US2022 / 027399, the disclosure of which is incorporated herein by reference.

[0220] [Table 17]

[0221] In some embodiments, the extracellular ActRII chimera has 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 removal of 1-9 amino acids from the N-terminus of any of the chimeras shown in Tables 15-17. The N-terminal truncation can remove 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 chimera ligand trap).

[0222] The extracellular ActRII chimeric ligand trap may further include a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension may add one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus of any of the chimeras shown in Tables 15-17. The C-terminal extension may correspond to a sequence from the same position in wild-type ActRIIA or ActRIIB. For example, the C-terminal extension that may be included in the extracellular ActRII chimeric ligand trap of the present invention is the amino acid sequence NP and the amino acid sequence NPVTPK (SEQ ID NO: 78), which correspond to the sequence found at the same position in wild-type ActRIIA.

[0223] In some embodiments, the extracellular ActRII chimeric ligand trap may further comprise a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) that may be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRII chimera via a linker or other covalent bond. An ActRII chimeric ligand trap comprising an extracellular ActRII chimera fused to an Fc domain monomer may form a dimer (e.g., a homodimer or heterodimer) due to interaction between the two Fc domain monomers that combine to form the Fc domain in the dimer.

[0224] Fc domain In some embodiments, the ActRII ligand trap described herein may comprise an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to increase serum half-life of the polypeptide. An ActRII ligand trap comprising 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) by interaction between two Fc domain monomers forming an Fc domain in the dimer. As conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain is a C-terminal end of an immunoglobulin. HIt comprises two Fc domain monomers that are dimerized by interactions between the three antibody constant domains. The Fc domain forms the minimal structure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV. In some embodiments, the Fc domain may be mutated to lack effector functions that are typical of a "dead" Fc domain. For example, the Fc domain may contain specific amino acid substitutions that are known to minimize interactions between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains 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)). Kabat numbering of amino acid residues can be determined for a given antibody by aligning the sequence of the antibody with the "standard" Kabat numbered sequence at the homology region. Furthermore, in some embodiments, the Fc domain does not induce any immune system related response. For example, the Fc domain in the dimer of ActRII ligand trap, which includes the extracellular portion of ActRIIA, ActRIIB, variants thereof, or chimeras thereof fused to an Fc domain monomer, can be modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. The sequence of the Fc domain monomer that can be fused to the extracellular ActRIIA variant is shown below (SEQ ID NO: 97).

[0225] THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions L12A, L13A, and G15A compared to the sequence of SEQ ID NO: 97. In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions D43A, K100A, and N212A compared to the sequence of SEQ ID NO: 97. In some embodiments, the terminal lysine is absent in an Fc domain monomer having a sequence of SEQ ID NO: 97. In some embodiments, the 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)) may be fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97) via conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof and the Fc domain monomer. The Fc domain monomer can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof.

[0226] In some embodiments, the ActRII ligand trap described herein may comprise an extracellular portion of ActRIIA, ActRIIB, variants thereof, or chimeras thereof fused to an Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain comprises a hinge domain. The Fc domain can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.

[0227] Methods for engineering Fc domains with reduced dimerization are known in the art. In some embodiments, one or more amino acids with large side chains (e.g., tyrosine or tryptophan) are replaced with C H 3-C H In another embodiment, one or more amino acids with small side chains (e.g., alanine, valine, or threonine) can be introduced into the C3 dimer interface to prevent dimer formation due to steric clashes. H 3-C H 3 dimer interface to remove favorable interactions. Amino acids with large or small side chains can be introduced into the C H Methods for introducing the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application 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.

[0228] In yet another embodiment, there is a C H 3-C H 3. C that constitutes the interface H One or more amino acid residues in the three domains are replaced with a positively charged amino acid residue (e.g., lysine, arginine, or histidine) or a negatively charged amino acid residue (e.g., aspartic acid or glutamic acid) such that the interaction is electrostatically unfavorable depending on the particular charged amino acid introduced. H Methods for introducing into the 3 domains to disfavor or prevent dimer formation are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication Nos. 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111.

[0229] In some embodiments of the invention, the Fc domain contains the following amino acid substitutions compared to the sequence of human IgG1: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, The Fc domain may comprise one or more of the following amino acid substitutions: 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. In some embodiments, the terminal lysine is absent from the Fc domain amino acid sequence. In one particular embodiment, the Fc domain comprises the amino acid substitution T366W compared to the sequence of human IgG1. The sequence of the Fc domain (wild type Fc domain) is shown below (SEQ ID NO: 84).

[0230] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK An exemplary sequence of a wild-type Fc domain lacking the terminal lysine is provided below (SEQ ID NO:79).

[0231] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Albumin-binding peptides In some embodiments, the ActRII ligand trap described herein may comprise an extracellular portion of ActRIIA, ActRIIB, its variant, or its chimera fused to a serum protein binding peptide. Binding to serum protein peptide may improve the pharmacokinetics of protein pharmaceuticals.

[0232] By way of example, albumin-binding peptides that can be used in the methods and compositions described herein are commonly known in the art. In one embodiment, the albumin-binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 83).

[0233] In the present invention, an albumin binding peptide can be linked to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof. In some embodiments, the albumin binding peptide is linked to the N-terminus or C-terminus of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof, either directly or via a linker.

[0234] In some embodiments, the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras 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)) may be fused to the N-terminus or C-terminus of an albumin-binding peptide (e.g., SEQ ID NO: 83) via 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 the ActRIIA, ActRIIB, variants thereof, or chimeras thereof and the albumin-binding peptide. Without being bound by theory, it is expected that the inclusion of an albumin-binding peptide in the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras thereof described herein may result in long-term retention of the therapeutic protein through its binding to serum albumin.

[0235] Fibronectin Domain In some embodiments, the ActRII ligand trap described herein may comprise an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to a fibronectin domain. Binding to the fibronectin domain may improve the pharmacokinetics of protein pharmaceuticals.

[0236] A fibronectin domain is, for example, a high molecular weight glycoprotein or fragment thereof of the extracellular matrix that binds to transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, a fibronectin domain is linked to the N-terminus or C-terminus (e.g., C-terminus) of the extracellular portion of ActRIIA, ActRIIB, its variants, or its chimeras described herein (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to increase the serum half-life of the extracellular portion of ActRIIA, ActRIIB, its variants, or its chimeras. The fibronectin domain can be linked to the N-terminus or C-terminus of the extracellular portion of ActRIIA, ActRIIB, its variants, or its chimeras, either directly or via a linker.

[0237] By way of example, fibronectin domains that can be used in the methods and compositions described herein are commonly known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain having amino acids 610 to 702 of the sequence of UniProt ID number P02751 (SEQ ID NO: 82 below).

[0238] GPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTST In another embodiment, the fibronectin domain is an adnectin protein.

[0239] In some embodiments, the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras 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)) may be fused to the N-terminus or C-terminus of a fibronectin domain (e.g., SEQ ID NO: 82) via 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 the ActRIIA, ActRIIB, variants thereof, or chimeras thereof and the fibronectin domain. Without being bound by theory, it is expected that the inclusion of a fibronectin domain in the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras thereof described herein may result in long-term retention of the therapeutic protein through its binding to integrins and extracellular matrix components such as collagen and fibrin.

[0240] Serum albumin In some embodiments, the ActRII ligand trap described herein may comprise an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to serum albumin. Binding to serum albumin may improve the pharmacokinetics of protein pharmaceuticals.

[0241] 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 serum proteins. It is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure required for proper distribution of body fluids between blood vessels and body tissues. In a preferred embodiment, the serum albumin is human serum albumin. In some embodiments of the present invention, human serum albumin is linked to the N-terminus or C-terminus (e.g., C-terminus) of the extracellular portion of ActRIIA, ActRIIB, its variant, or its chimera 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)) to increase the serum half-life of the extracellular portion of ActRIIA, ActRIIB, its variant, or its chimera. Human serum albumin can be linked, either directly or via a linker, to the N-terminus or C-terminus of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or chimera thereof.

[0242] By way of example, serum albumin that can be used in the methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID number P02768 (SEQ ID NO: 81 below).

[0243] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEP ERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVV LNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL In some embodiments, the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras 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)) may be fused to the N-terminus or C-terminus of human serum albumin (e.g., SEQ ID NO: 81) via 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 the ActRIIA, ActRIIB, variants thereof, or chimeras thereof and human serum albumin. Without being bound by theory, it is expected that the inclusion of human serum albumin in the extracellular portion of the ActRIIA, ActRIIB, variants thereof, or chimeras thereof described herein may result in long-term retention of the therapeutic protein.

[0244] Linker The ActRII ligand traps described herein may include an extracellular portion of an ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused via a linker to a moiety (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 moiety enhances the stability of the polypeptide. Exemplary moieties include an Fc domain monomer, an Fc domain, an albumin binding peptide, a fibronectin domain, or 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), an Fc domain (e.g., SEQ ID NO: 84 or SEQ ID NO: 79), an albumin-binding peptide (e.g., SEQ ID NO: 83), a fibronectin domain (e.g., SEQ ID NO: 82), or human serum albumin (e.g., SEQ ID NO: 81)) and ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be an amino acid spacer containing 1 to 200 amino acids. Suitable peptide spacers are known in the art, and these include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the spacer can include a 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) motif, e.g., multiple chief or repeat motifs.In some embodiments, the spacer can include 2-12 amino acids including a GA or GS motif, 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 GSGSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer can include 3-12 amino acids including a GGA or GGS motif, 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). Further, in some embodiments, the spacer can include 4 to 12 amino acids including the motifs 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, the spacer can include the motifs GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGAGGGGAGGGGA (SEQ ID NO: 128) and GGGSGGGGSGGGGGS (SEQ ID NO: 129).In some embodiments of the invention, the amino acid spacer between a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) and the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be 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).

[0245] In some embodiments, the spacer can also include amino acids other than glycine, alanine, and serine, such as AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAR (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), and GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 88). In some embodiments, the spacer can include a motif, such as a multiple motif or a repeated motif, of EAAAK (SEQ ID NO: 87). In some embodiments, the spacer is (XP) n wherein X can be any amino acid (e.g., A, K, or E) and n is 1-5, and can include a proline-rich sequence and a motif, e.g., a multiple motif or a repeated motif, of PAPAP (SEQ ID NO: 86).

[0246] 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.

[0247] In some embodiments, the linker between a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), an Fc domain (e.g., SEQ ID NO: 84 or SEQ ID NO: 79), an albumin binding peptide (e.g., SEQ ID NO: 83), a fibronectin domain (e.g., SEQ ID NO: 82), or human serum albumin (e.g., SEQ ID NO: 81)) and an ActRIIA, ActRIIB, variant thereof, or 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)) is an amino acid spacer having the sequence GGG. For example, an ActRIIA ligand trap of the present invention can include an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 6-72) fused to an Fc domain (e.g., SEQ ID NO: 79) via a GGG linker. An exemplary polypeptide comprising an ActRIIA variant of SEQ ID NO: 69, a GGG linker, and an Fc domain lacking a terminal lysine (SEQ ID NO: 79) is provided below (SEQ ID NO: 80).

[0248] GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Vectors, host cells, and protein production The ActRII signal transduction inhibitor of the present invention can be produced from a host cell. A host cell refers to a vehicle that contains the cellular components, e.g., organelles, necessary for expressing the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acid can be contained in a nucleic acid vector that can be introduced into a host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The selection of the nucleic acid vector depends in part on the host cell used. In general, the preferred host cell is either of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0249] Nucleic Acid Vector Constructs and Host Cells Nucleic acid sequences encoding the amino acid sequence of the polypeptide of the present invention (i.e., ActRII signaling inhibitors) can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptide of the present invention can be obtained using standard techniques, such as gene synthesis. Alternatively, in the case of production of ActRII ligand traps, nucleic acid molecules encoding the wild-type portion of extracellular ActRIIA or ActRIIB can be mutated to contain specific amino acid substitutions using techniques standard in the art, such as QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0250] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector that can replicate and express the nucleic acid molecule in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector can contain various components that can be adjusted and optimized for compatibility with a specific host cell. For example, vector components can include, but are not limited to, a replication origin, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding a protein of interest, and a transcription termination sequence.

[0251] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells can also be used as hosts of the present invention. Examples of E. coli strains include E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC® 31,537, E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608), but are not limited thereto. Various host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of expressed polypeptides. The above expression vectors can be introduced into suitable host cells using techniques conventional in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector has been introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media, modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences.Methods for expressing therapeutic proteins are known in the art, see, e.g., Paulina Balbas, 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.

[0252] Protein Production, Recovery, and Purification The host cells used to produce the polypeptides of the invention may be grown in a medium suitable for culturing the selected host cells known in the art. Examples of suitable media for mammalian host cells include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria Broth (LB) containing necessary supplements such as a selection agent, e.g., ampicillin. The host cells may be cultured at a suitable temperature, e.g., about 20° C. to about 39° C., e.g., 25° C. to about 37° C., preferably 37° C., and in a suitable CO2 medium. 2 The cells are cultured at a pH level of, for example, 5-10%. The pH of the medium is generally about 6.8-7.4, for example, 7.0, depending mainly on the host organism. When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter.

[0253] In some embodiments, depending on the expression vector and host cell used, the expressed protein may be secreted from the host cell (e.g., mammalian host cell) into the cell culture medium. Protein recovery may include filtering the cell culture medium to remove cellular debris. The protein may be further purified. The polypeptides of the invention may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential lysis, or any other standard technique for protein purification. For example, the protein may be isolated and purified by appropriately selecting an affinity column, such as a Protein A column (e.g., POROS Protein A chromatography), in combination with a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis procedures.

[0254] In other embodiments, the host cells can be disrupted, for example, by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, the cell debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be conjugated to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His tag), which binds to nickel-functionalized agarose affinity columns 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 the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0255] Alternatively, a polypeptide of the invention can be produced by cells of a subject (e.g., a human) by administering a vector (e.g., a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector such as Modified Vaccinia Ankara (MVA), an adeno-associated virus vector, and an alphavirus vector)) comprising a nucleic acid molecule encoding a polypeptide of the invention, e.g., in the context of gene therapy. The vector, once inside the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), promotes expression of the polypeptide, which is then secreted from the cells. If treatment of a disease or disorder is the desired outcome, no further measures may be necessary. If collection of protein is desired, blood can be collected from the subject and the protein purified from the blood by methods known in the art.

[0256] Pharmaceutical Compositions and Preparations The invention features pharmaceutical compositions comprising an ActRII signaling inhibitor, such as an ActRII ligand trap comprising an extracellular portion of a polypeptide described herein (e.g., 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)). In some embodiments, the pharmaceutical compositions of the invention comprise, as a therapeutic protein, an ActRII ligand trap comprising an extracellular ActRIIA variant having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). In some embodiments, the pharmaceutical compositions of the invention comprise, as a therapeutic protein, an ActRII ligand trap comprising a moiety (e.g., an Fc domain monomer, or a dimer thereof, Fc domain monomer, or a dimer thereof, Fc domain monomer, or a dimer thereof). The ActRII ligand trap includes an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a polypeptide of the present invention (e.g., a polypeptide of the present invention), an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof ... an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, the pharmaceutical composition of

[0257] Acceptable carriers and excipients in pharmaceutical compositions are non-toxic to recipients at the dosages and concentrations used. 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, octadecyldimethylbenzylammonium 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. The pharmaceutical composition of the present invention can be administered parenterally in the form of an injectable formulation. The pharmaceutical composition for injection can be formulated using a sterile solution or any pharma- ceutical acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), alpha-Modified Eagle Medium (alpha-MEM), F-12 medium). Formulation methods are known in the art, see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor & Francis Group, CRC Press (2015).

[0258] The pharmaceutical compositions of the invention may be formulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the invention may also be formulated 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 22 ndPharmaceutical compositions to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0259] The pharmaceutical composition of the present invention may be prepared as a sustained release formulation. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide of the present invention. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-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 allow the release of molecules over several months, e.g., 1-6 months, while other formulations release the pharmaceutical composition of the present invention over shorter periods, e.g., days to weeks.

[0260] The pharmaceutical composition may be formed into a unit dosage form as needed. The amount of active ingredient, for example, the polypeptide of the present invention, contained in the pharmaceutical preparation is such that a suitable dose within the specified range (for example, a dose within the range of 0.01 to 100 mg / kg body weight) is provided.

[0261] 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 embedded.When hydrodynamic injection is used as a delivery method, the pharmaceutical composition containing the nucleic acid molecule encoding the polypeptide described herein or the vector (e.g., viral vector) containing the nucleic acid molecule is rapidly delivered intravenously in a large volume of liquid.Vector that can be used as an in vivo gene delivery vehicle includes, but is not limited to, retroviral vector, adenoviral vector, poxvirus vector (e.g., vaccinia virus vector such as modified vaccinia Ankara), adeno-associated virus vector, and alphavirus vector.

[0262] Route, Dosage, and Administration The pharmaceutical composition comprising the polypeptide of the present invention as a therapeutic protein can be formulated for example for intravenous, parenteral, subcutaneous, intramuscular, intraarterial, intrathecal 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, for example, ASHP Handbook on Injectable Drugs, Toissel, 18th ed.(2014).

[0263] In some embodiments, pharmaceutical compositions comprising nucleic acid molecules encoding the polypeptides of the present invention or vectors comprising such nucleic acid molecules can be administered by gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors such as modified vaccinia Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, mRNA molecules encoding the polypeptides of the present invention can be administered directly to the subject.

[0264] In some embodiments of the present invention, nucleic acid molecules encoding the polypeptides described herein or vectors containing such nucleic acid molecules may be administered using a hydrodynamic injection platform. In hydrodynamic injection, the nucleic acid molecules encoding the polypeptides described herein are placed 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 volume of liquid intravenously. Hydrodynamic injection uses controlled hydrodynamic pressure in the vein to enhance cell permeability, such that the pressure increase caused by the rapid injection of a large volume of liquid results in fluid and plasmid leakage from the vein. Expression of the nucleic acid molecule is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding the polypeptides described herein may be administered using hydrodynamic injection.

[0265] The dosage of the pharmaceutical composition of the invention will depend on factors including the route of administration, the disease being treated, and the physical characteristics, such as age, weight, and general health of the subject. The pharmaceutical compositions of the present invention comprise a dosage of an ActRII signaling inhibitor of the present invention in the range of 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), in 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 can be adapted by the physician according to conventional factors such as the degree of the subject's disease and different parameters.

[0266] The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in an amount therapeutically effective to result in improvement or remediation of symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, for example, intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Generally, the therapeutic protein is administered at 0.1-100 mg / kg, for example, 0.5-50 mg / kg. The pharmaceutical compositions comprising the polypeptides of the invention may be administered one or more times (e.g., 1-10 or more times) to a subject in need thereof, for example, daily, weekly, biweekly, every four weeks, monthly, bimonthly, quarterly, biennially, annually, or as medically indicated. In some embodiments, the pharmaceutical compositions comprising the polypeptides of the invention may be administered weekly, biweekly, every four weeks, monthly, bimonthly, or quarterly to a subject in need thereof. Dosages may be provided in either single or multiple dosage regimens. The interval between doses may shorten as the condition improves or may lengthen as the patient's health deteriorates.

[0267] Treatment method The ActRII signaling inhibitors described herein (e.g., activin A antibody, myostatin antibody, activin B antibody, GDF-11 antibody, ActRII antibody, or ActRII ligand trap) can be used to treat a subject undergoing treatment with a cytopenia-associated myelofibrosis therapy. In some embodiments, the subject has cytopenia (e.g., anemia, thrombocytopenia, and / or neutropenia) (e.g., the subject has already developed cytopenia or has been identified as having cytopenia prior to treatment with an ActRII signaling inhibitor described herein). In some embodiments, the subject has not yet developed cytopenia or has not been identified as having cytopenia when treatment with an ActRII signaling inhibitor is initiated. In some embodiments, the subject is undergoing treatment with a thrombocytopenia-associated 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-risk or high-risk myelofibrosis. In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance score of 2 or less. In some embodiments, the subject is being treated with a cytopenia-associated myelofibrosis treatment for polycythemia vera. In some embodiments, the subject is being treated with a cytopenia-associated myelofibrosis treatment for steroid-refractory acute graft-versus-host disease. In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis treatment has anemia. Anemia is defined as a hemoglobin of 10 g / dL or less during screening or while receiving an RBC transfusion. In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis treatment has thrombocytopenia. In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis treatment has both anemia and thrombocytopenia. In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis treatment has neutropenia. In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis treatment has anemia and neutropenia.In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis therapy has thrombocytopenia and neutropenia. In some embodiments, the subject being treated with a cytopenia-associated myelofibrosis therapy has anemia, thrombocytopenia, ... has cytopenia-associated bone marrow fibrosis for at least 8 weeks (e.g., 8 weeks or more, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 weeks or more, or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more) prior to co-administration of an ActRII signaling inhibitor described herein. have been treated with a myelofibrosis therapy and have been taking a stable dose of a cytopenia-associated myelofibrosis therapy for at least 4 weeks (e.g., for more than 4 weeks, e.g., for more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 weeks, or for more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months); In some embodiments, the subject has been treated with a cytopenia-associated myelofibrosis therapy for at least 8 weeks and less than 6 months (e.g., 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, and has been taking a stable dose of a cytopenia-associated 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). In some embodiments, the subject has been treated with a cytopenia-associated myelofibrosis therapy for less than 8 weeks (e.g., 7, 6, 5, 4, 3, 2, 1 week or less) prior to co-administration of an ActRII signaling inhibitor described herein.In some embodiments, the cytopenia-associated myelofibrosis treatment and treatment with the ActRII signaling inhibitor are initiated simultaneously (e.g., the subject begins treatment with both agents at about the same time, e.g., begins treatment with both agents on the same day, week, or month). In some embodiments, the subject is identified as having a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with the cytopenia-associated myelofibrosis treatment prior to co-administration of an ActRII signaling inhibitor described herein. In some embodiments, the method includes identifying a subject (e.g., by assessing red blood cell, hemoglobin, hematocrit, platelet, and / or neutrophil levels) as having a cytopenia (e.g., anemia, thrombocytopenia, or neutropenia) associated with the cytopenia-associated myelofibrosis treatment prior to co-administration of an ActRII signaling inhibitor described herein. The method may further include evaluating red blood cell, hematocrit, reticulocyte, platelet, and / or neutrophil levels after administration of an ActRII signaling inhibitor described herein (e.g., by CBC count, 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 initiation of treatment with an ActRII signaling inhibitor described herein). In some embodiments, the subject is not receiving concomitant treatment with erythropoiesis stimulating agents (ESAs), granulocyte colony stimulating factors (G-CSFs), granulocyte-macrophage colony stimulating factors (GM-CSFs), thrombopoietin agonists (TPOs), immunomodulatory imide drugs (IMiDs, e.g., thalidomide, pomalidomide, lenalidomide), interferons, hydroxyurea, danazol, or steroids (excluding up to 10 mg / day of prednisone or corticosteroid equivalents). In some embodiments, the subject has not been previously treated with luspatercept, sotatercept, or other TGF-β inhibitors.

[0268] In some embodiments, the methods described herein increase hemoglobin levels, increase hematocrit, increase red blood cell count, increase red blood cell volume, increase red blood cell mass, increase reticulocytes, increase proerythroblasts, increase or induce red blood cell formation or production, increase maturation and / or differentiation of erythroid progenitor cells (e.g., early or late (e.g., terminal) progenitor cells, e.g., early erythroid progenitor cells, e.g., burst forming units-erythroid (BFU-E) and / or red blood cells, or increase the maturation and / or differentiation of erythroid progenitor cells (e.g., early or late (e.g., terminal) progenitor cells, e.g., early erythroid ... increasing maturation and / or differentiation of colony forming units (CFU-E), e.g., BFU-E and / or CFU-E, into proerythroblasts, reticulocytes, or erythrocytes, e.g., increasing proerythroblast and / or reticulocyte numbers), increasing late erythroid precursor maturation (e.g., terminal maturation, e.g., maturation of reticulocytes into erythrocytes, or maturation of erythroblasts into reticulocytes and / or erythrocytes), mobilizing early progenitor cells into the erythroid lineage, increasing early erythroid precursors and / or progenitor cell numbers (e.g., early progenitor proliferate the erythroid population to provide a continuous supply of precursors to replenish polychromatic erythroblasts and allow for a continuous supply of mature reticulocytes), promote the progression of erythroid precursors and / or progenitor cells through erythropoiesis, reduce the accumulation of erythroid progenitor cells (e.g., by stimulating progenitor cells to mature), increase platelet levels (e.g., increasing platelet count), increase or induce megakaryocyte differentiation and / or maturation (e.g., terminal maturation of platelet progenitor cells into platelets to produce platelets), reduce the accumulation of platelet progenitor cells (e.g., by stimulating progenitor cells to mature), increase megakaryocyte progenitor cells (e.g., increasing megakaryocyte progenitor cell renewal), increase platelet progenitor cells, promote or increase platelet formation or production, increase neutrophil levels (e.g., increasing neutrophil count), increase or induce differentiation and / or maturation of progenitor cells (e.g., bone marrow progenitor cells, myeloblasts, or myelocytes) into neutrophils, and / or increase or induce neutrophil formation or production in a subject. In some embodiments, the methods described herein increase the rate of recovery from thrombocytopenia.These changes may be observed in subjects treated with an ActRII signaling inhibitor as described herein compared to measurements obtained prior to treatment or compared to measurements obtained from subjects treated with only cytopenia-associated myelofibrosis treatment. In some embodiments, the methods described herein improve or restore hematopoiesis in bone marrow, reduce or reverse reticulin and / or collagen deposition, or reverse bone changes associated with myelofibrosis. In some embodiments, the methods described herein reduce or ameliorate megakaryocyte dysfunction (e.g., megakaryocyte dysfunction in bone marrow), which may prevent or reduce inflammation / fibrosis, restore hematopoiesis in bone marrow, and treat cytopenias due to myelofibrosis and cytopenias due to JAK inhibitor treatment. In some embodiments, the methods described herein reduce or ameliorate hepatosplenomegaly or splenomegaly (e.g., reduce splenic volume and / or splenic extramedullary hematopoiesis) and symptoms thereof. In some embodiments, the methods described herein reduce myelofibrosis and alleviate symptoms caused by bone marrow loss. In some embodiments, the methods described herein slow or reduce the progression of myelofibrosis. In some embodiments, the methods described herein improve or ameliorate the attenuated bone resorption and bone sclerosis in patients with myelofibrosis. In some embodiments, the methods described herein improve fibrosis, bone histology, spleen size (e.g., reduce spleen size), myelofibrosis symptoms, myelofibrosis, 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 cytopenias (e.g., anemia, thrombocytopenia, and / or neutropenia) caused by cytopenia-associated myelofibrosis treatment, and reverse the decrease in red blood cells, platelets, and / or neutrophils induced by cytopenia-associated myelofibrosis treatment. In some embodiments, the methods described herein reduce bleeding events. In some embodiments, the methods described herein reduce infection.

[0269] In some embodiments, treatment with the methods described herein results in a mean hemoglobin increase of 1.5 g / dL or 2.0 g / dL or more from baseline or pre-treatment measurements for 12 or more consecutive weeks, e.g., 12, 14, 16, 18, 20, 22, 24, 26 weeks, 1 year, 2 years or more, during treatment with an ActRII signaling inhibitor described herein, e.g., the first 24 or 52 weeks of treatment of a transfusion-independent subject with a method described herein. In some embodiments, treatment with the methods described herein results in a reduction of 1 or more in the Short Fatigue Scale score from baseline within the first 24 or 52 weeks of treatment of a transfusion-independent subject with a method described herein. In some embodiments, the methods described herein reduce the transfusion requirement of a subject requiring RBC transfusions, e.g., a subject with anemia (e.g., reduce transfusion burden, e.g., the subject no longer requires transfusions or the subject requires transfusions less frequently than before treatment with the compositions and methods described herein). In some embodiments, treatment with the methods described herein reduces the number of RBC transfusions from baseline pre-treatment measurements (e.g., measurements taken over a 12-week period immediately prior to initiation of treatment with an ActRII signaling inhibitor described herein) for a period of 12 or more consecutive weeks, e.g., 12, 14, 16, 18, 20, 22, 24, 26 weeks, 1 year, 2 years or more, during treatment with an ActRII signaling inhibitor described herein, e.g., during the first 24 or 52 weeks of treatment with a method described herein.In some embodiments, the compositions and methods described herein promote transfusion independence (e.g., a subject who required one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) RBC units over the 12 weeks immediately prior to initiating treatment with an ActRII signaling inhibitor described herein does not require transfusions for 12 or more consecutive weeks, e.g., 12, 14, 16, 18, 20, 22, 24, 26 weeks, 1 year, 2 years or more, during treatment immediately prior to initiating treatment with an ActRII signaling inhibitor described herein, e.g., during the first 24 or 52 weeks of treatment with a method described herein). Concomitant treatment of anemia with RBC transfusions is recommended when hemoglobin is less than 8.0 g / dL and when Hgb is 8.0 g / dL or greater and associated with symptom(s) of anemia (e.g., hemodynamic or pulmonary failure requiring treatment) or a comorbidity warranting an Hgb threshold of 8.0 g / dL or greater. A complete blood count (CBC) can be taken to assess the subject's response to treatment with the compositions described herein, and hemoglobin levels can be examined 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 can increase. In some embodiments, treatment with the methods described herein results in an improvement in Myelofibrosis Symptom Assessment Form Total Symptom Score (MF-SAF-TSS) of 50% or more from baseline (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more from baseline), for example, after 24 weeks or 52 weeks of treatment with the methods described herein. In some embodiments, treatment with the methods described herein results in a reduction in spleen volume of 35% or more from baseline (e.g., at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more from baseline) as measured by computed tomography, e.g., after 24 weeks or 52 weeks of treatment with the methods described herein.In some embodiments, the compositions and methods described herein delay or inhibit progression to acute myeloid leukemia (AML) (>20% bone marrow blasts) and / or accelerated MF (>10% bone marrow blasts), e.g., by 24 or 52 weeks of treatment with the methods described herein. In some embodiments, treatment with the methods described herein reduces or inhibits progression to acute myeloid leukemia (AML) (>20% bone marrow blasts) by 30×10 from baseline over 12 weeks or more, e.g., 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 (without platelet transfusions), e.g., by 24 or 52 weeks of treatment with the methods described herein. 9In some embodiments, treatment with the methods described herein reduces episodes of anemia, neutropenia, and thrombocytopenia of grade 1 or higher. In some embodiments, treatment with the methods described herein allows a subject to maintain dose intensity of cytopenia-associated myelofibrosis treatment. In some embodiments, treatment with the methods described herein improves tolerability or adherence to thrombocytopenia-associated myelofibrosis treatment, e.g., a subject can continue thrombocytopenia-associated myelofibrosis treatment for 12 weeks, 24 weeks, 52 weeks, or more with combination treatment with an ActRII signaling inhibitor described herein. In some embodiments, treatment with the methods described herein reduces bone sclerosis from baseline, as assessed using CT, e.g., with 24 weeks or 52 weeks of treatment as described herein. In some embodiments, treatment with the methods described herein results in a reduction in Patient-Reported Outcomes Measurement Information System (PROMIS) score or BFI score from baseline, e.g., with 24 weeks or 52 weeks of treatment as described herein. In some embodiments, treatment with the methods described herein slows or reduces the progression of myelofibrosis or improves (e.g., reverses) myelofibrosis. For example, treatment with the methods described herein may result in an improvement in myelofibrosis grade from baseline or prevent a worsening of myelofibrosis grade, for example, with 24 weeks or 52 weeks of treatment as described herein. Treatment with the methods described herein may also increase red blood cell parameters, such as reticulocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and reticulocyte hemoglobin, and / or biomarkers of blood cell production, such as erythropoietin (EPO) and thrombopoietin (TPO) levels. In some embodiments, treatment with the methods described herein increases biomarkers of bone metabolism, such as bone-specific alkaline phosphatase (BSAP) and serum C-telopeptide of collagen type I (CTX), compared to baseline.In some embodiments, treatment with the methods described herein reduces the incidence of myelofibrosis-associated molecular and cytogenetic abnormalities over the treatment period. Treatment with the methods described herein may also result in changes in biomarkers of iron metabolism (e.g., serum iron, ferritin, transferrin, transferrin saturation, total iron binding capacity, soluble transferrin receptor levels, and hepcidin), iron chelator doses, and cytokine levels compared to baseline with 24 or 52 weeks of treatment as described herein.

[0270] In some embodiments, the methods described herein do not cause any vascular complications, such as increased vascular permeability or leakage, in a subject. In some embodiments, the ActRII signaling inhibitor co-administered with the cytopenia-associated myelofibrosis treatment is an ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, the ActRIIA ligand trap comprising an extracellular ActRIIA variant is administered at a dose of 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.7 In a more specific embodiment, the therapeutic protein is administered at a dosage ranging from about 0.5, 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, from about 0.1 to about 30 mg / kg, and in a more specific embodiment, from about 0.3 to about 30 mg / kg. In any of the methods described herein, an ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs: 6-71)) further comprising 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 a therapeutic protein. In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) formed by the interaction of two Fc domain monomers, each fused to a polypeptide comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), may be used as a therapeutic protein.In any of the methods described herein, an ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety (e.g., an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be used as a therapeutic protein. A nucleic acid encoding a polypeptide described herein, or a vector comprising the nucleic acid, may also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector may be administered as part of a pharmaceutical composition.

[0271] Compositions that can be administered to a subject according to the methods described herein are provided below in Tables 18-21. Combination therapy The ActRII signaling inhibitor described herein is administered to a subject in combination with a cytopenia-associated myelofibrosis treatment. The cytopenia-associated myelofibrosis treatment can be, for example, ruxolitinib (JAKAFI® / JAKAVI®), fedratinib (INREBIC®), pacritinib (VONJO™), or imetelstat. The cytopenia-associated myelofibrosis treatment can be administered simultaneously with the ActRII signaling inhibitor (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes). These agents can also be administered simultaneously by co-formulation. The ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can also be administered sequentially, which allows the two actions to overlap, and their combined effect is greater in reducing symptoms or other parameters associated with the disorder than observed when one agent or treatment is delivered alone or in the absence of the other agent or treatment. The effect of the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment may be partially additive, completely additive, or more than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each of the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can be performed by any suitable route, including, but not limited to, oral, intravenous, intramuscular, topical, and direct absorption through mucosal tissue. The ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can be administered by the same route or by different routes. For example, the ActRII signaling inhibitor may be administered by subcutaneous (e.g., in the case of an ActRII ligand trap) or intravenous (e.g., in the case of activin A, activin B, myostatin, GDF-11, or ActRII antibody) injection or infusion, while the cytopenia-associated myelofibrosis treatment may be administered by oral (in the case of ruxolitinib, fedratinib, or pacritinib) or intravenous (in the case of imetelstat) injection or infusion.The ActRII signaling inhibitor can be administered immediately before or immediately after the thrombocytopenia associated myelofibrosis treatment, 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, up to 14 hours, up to 16 hours, up to 17 hours, up to 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 before or after the thrombocytopenia associated myelofibrosis treatment, or up to 1-7 days, 1-14 days, 1-21 days, or 1-30 days before or after the thrombocytopenia associated myelofibrosis treatment. In some embodiments, the ActRII signaling inhibitor and the cytopenia associated myelofibrosis treatment are administered at different frequencies. For example, the ActRII signaling inhibitor can be administered once a week, once every two weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or once every six months, and the cytopenia-associated myelofibrosis treatment can be administered once or twice a day (e.g., in the case of ruxolitinib, fedratinib, or pacritinib). In some embodiments, the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment are administered at the same or similar frequency. For example, both the ActRII signaling inhibitor and the cytopenia-associated myelofibrosis treatment can be administered once a week, once every two weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or once every six months (e.g., in the case of imetelstat, in the case of cytopenia-associated myelofibrosis treatment).

[0272] In some embodiments, the cytopenia-associated myelofibrosis treatment is administered as directed on the label. For example, ruxolitinib is administered to patients with myelofibrosis and a baseline myelofibrosis of 200×10 9 A starting dose of 20 mg orally twice daily is given for subjects with myelofibrosis and platelets >100 × 10 / L at baseline. 9 / L~200×10 9 / L, have myelofibrosis and have a platelet count of 50 × 10 at baseline 9 / L~100×109 For subjects with less than 1 / L of platelets, 5 mg is administered orally twice daily, for polycythemia vera, 10 mg is administered orally twice daily, or for acute graft-versus-host disease, 5 mg is administered orally twice daily, which can be increased to 10 mg twice daily after at least 3 days of treatment. In some embodiments, ruxolitinib is administered at a dose of 10 mg / day to 50 mg / day (e.g., 10 mg / day, 15 mg / day, 20 mg / day, 25 mg / day, 30 mg / day, 35 mg / day, 40 mg / day, 45 mg / day, or 50 mg / day). When administered alone, dosing may need to be reduced or discontinued due to the development of cytopenias, but when administered in combination with an ActRII signaling inhibitor, subjects may be able to continue taking the same dose of ruxolitinib with little or no discontinuation of treatment, and may be able to receive higher doses of ruxolitinib (e.g., 5 mg or more, e.g., 5 mg, 10 mg, or 15 mg) compared to the dose of ruxolitinib when administered alone. Fedratinib is administered in subjects with myelofibrosis (e.g., with intermediate-2 risk or high-risk primary or secondary myelofibrosis and ≥ 50×10 at baseline). 9 Pacritinib may be taken at a dose of 400 mg or less once a day, for example, 400 mg, 300 mg, 200 mg, or 100 mg once a day, by subjects with platelets greater than 500 mg / L. When administered alone, it may be necessary to reduce or discontinue dosing due to the development of cytopenias, but when administered in combination with an ActRII signaling inhibitor, subjects may be able to continue taking the same dose of fedratinib with little or no discontinuation of treatment. Pacritinib may be taken at a dose of 400 mg or less once a day, for example, 400 mg, 300 mg, 200 mg, or 100 mg once a day, by subjects with myelofibrosis (for example, 50×10 at baseline). 9For subjects with intermediate- or high-risk primary or secondary (post-polycythemia vera or post-essential thrombocytosis) myelofibrosis with a platelet count less than 1 / L, pacritinib may be taken at a dose of 200 mg twice daily, which may be reduced to 100 mg twice daily or 100 mg once daily if a dose modification is required due to adverse reactions. When administered alone, dosing may need to be reduced or discontinued due to the development of cytopenias, but when administered in combination with an ActRII signaling inhibitor, subjects may be able to continue taking the same dose of pacritinib with little or no discontinuation of treatment. Imetelstat may be administered by intravenous infusion at a dose of about 1.0 mg / kg to about 50 mg / kg (e.g., 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0, 9.4, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, or 50.0 mg / kg) once a week, once every two weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or once every six months. When administered alone, dosing may need to be reduced or discontinued due to the onset of cytopenias, whereas when administered in combination with an ActRII signaling inhibitor, the subject may be able to continue taking the same dose of imetelstat with little or no cessation of treatment, may be able to receive higher doses of imetelstat (e.g., 1.0 mg / kg or more, e.g., 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg or more) or may be able to receive imetelstat treatment less frequently compared to the dose when administered alone.The ActRII signaling inhibitor is about 0.01 to about 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 15 In a specific embodiment, the compound can be administered at a dose of about 0, 200, 250, 300, 350, 400, 450, or 500 mg / kg, and in a more specific embodiment, at a dose of about 0.1 to about 30 mg / kg, and in a more specific embodiment, at a dose of about 0.3 to about 30 mg / kg, by subcutaneous or intravenous injection once a week, once every two weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or once every six months, or once a year.

[0273] In some embodiments, the combination therapy of an ActRII signaling inhibitor with a cytopenia-associated myelofibrosis treatment reduces adverse reactions associated with the cytopenia-associated myelofibrosis treatment, such as the development of anemia, thrombocytopenia, and / or neutropenia, which may lead to treatment interruption and discontinuation. In some embodiments, the combination therapy reduces or ameliorates anemia, thrombocytopenia, and / or neutropenia, or reduces the number of episodes of one or more of these cytopenias. In some embodiments, the combination therapy improves adherence to treatment with the thrombocytopenia-associated myelofibrosis treatment (e.g., the subject can continue treatment for a longer period of time), improves tolerability of the cytopenia-associated myelofibrosis treatment (e.g., the subject can maintain the same dose or increase the dose of the cytopenia-associated myelofibrosis treatment), reduces transfusion burden, reduces bleeding events, reduces infections, or reduces treatment interruption or discontinuation with the cytopenia-associated myelofibrosis treatment.

[0274] kit The ActRII signaling inhibitor and cytopenia-associated myelofibrosis treatment described herein may be provided in a kit for use in treating myelofibrosis. Each agent may be provided in a unit dosage form, optionally in a pharma- ceutically acceptable excipient (e.g., saline), in an amount sufficient to treat myelofibrosis. The kit may further include a package insert instructing a user of the kit, e.g., a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the ActRII signaling inhibitor or cytopenia-associated myelofibrosis treatment.

[0275] [Table 18-1]

[0276] [Table 18-2]

[0277] [Table 18-3]

[0278] [Table 18-4]

[0279] [Table 18-5]

[0280] [Table 18-6]

[0281] [Table 19-1]

[0282]

Table 19-2

[0283]

Table 19-3

[0284]

Table 19-4

[0285]

Table 19-5

[0286]

Table 19-6

[0287]

Table 19-7

[0288]

Table 20-1

[0289]

Table 20-2

[0290]

Table 20-3

[0291]

Table 20-4

[0292]

Table 20-5

[0293]

Table 20-6

[0294]

Table 20-7

[0295]

Table 20-8

[0296]

Table 20-9

[0297]

Table 20-10

[0298]

Table 21-1

[0299]

Table 21-2

[0300]

Table 21-3

[0301]

Table 21-4

[0302]

Table 21-5

[0303] [Table 21-6] EXAMPLES

[0304] 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, and it will be understood that by their illustrative nature, other procedures, methods, or techniques known to those skilled in the art may be substituted therefor.

[0305] Example 1 - Effect of ActRIIA / B-mFc on platelets Eleven-week-old C57Bl / 6 mice were administered either TBS (vehicle) or ActRIIA / B-mFc (10 mg / kg) by intraperitoneal (IP) administration. Whole blood was sampled 12 hours after administration, and platelet counts were determined using a veterinary hematology analyzer (Heska Element HT5). Mice were then euthanized, and bone marrow was extracted from the femur. Bone marrow cells were stained with antibodies against lineage (Perc-Cy5), sca1 (BV525), cKit (Alexa750), CD41 (APC), and CD150 (Pcy7) and analyzed by flow cytometer (Cytoflex, Beckman coulter). Megakaryocytic progenitor cells were gated into Lin-, sca1-, ckit+, CD150+, and CD41+ cells.

[0306] FIG. 1 shows the effect of ActRIIA / B-mFc on platelet production. A single dose of ActRIIA / B-mFc increased circulating platelet counts and bone marrow megakaryocyte progenitor cells within 12 hours of administration. The timing of the effect on platelets suggests a direct effect of ActRIIA / B-mFc on the final maturation of platelet progenitor cells to platelets, and the megakaryocyte progenitor cell data demonstrate that ActRIIA / B-mFc affects an early step in the platelet formation process. Data are expressed as mean ± standard error of the mean. Statistical analysis was performed using Student's T-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0307] Example 2 - Effect of ActRIIA / B-mFc on megakaryocyte maturation C57Bl / 6 mice aged 11 weeks were administered either TBS (vehicle) or ActRIIA / B-mFc (10 mg / kg) by IP administration. After 12 and 24 hours, mice were euthanized and bone marrow was extracted from femurs. Bone marrow cells were fixed with ethanol and subsequently stained with propidium iodide (PI) and anti-CD41 (FITC, Efferet) antibodies in parallel with RNAse treatment. Samples were analyzed with a flow cytometer (Cytoflex, Beckman coulter). The ploidy of CD41+ nucleated cells (PI+ cells) was analyzed. The graph represents the % of cells at each ploidy stage. N=3 for 12 hours and N=2 for 24 hours. For CD41+ cells at 12 hours, t-test was performed for statistical analysis. Data are presented as mean ± standard error of the mean.

[0308] Figure 2 shows that ActRIIA / B-mFc treatment exhibits a direct effect on megakaryocyte differentiation and maturation, as shown by an increase in the number of CD41+ megakaryocyte progenitors at 12 hours and an increase in the number of polyploid megakaryocytes at 24 hours after treatment. These data show an early effect on progenitor cells in the platelet production pathway, indicate that ActRIIA / B-mFc increased differentiation of megakaryocyte precursors and induced a later maturation stage, and demonstrate that ActRIIA / B-mFc treatment results in more megakaryocytes that can be primed for further platelet progenitor production.

[0309] Example 3 - Effect of ActRIIA / B-mFc on platelet recovery after depletion Twelve-week-old mice were treated with either anti-GP1bα (0.08 mg / kg, Efferet) or IgG control. Four days after treatment, the anti-GP1bα-treated group was further divided to receive either vehicle or ActRIIA / B-mFc (7.5 mg / kg) treatment. Platelets were measured at the indicated time points after anti-GP1bα dosing. Ten days after treatment, mice were euthanized and bone marrow cells were harvested. Bone marrow cells were fixed with ice-cold 100% ethanol and stained with propidium iodide (PI) (200 μg / mL, Sigma-Aldrich) and anti-CD41 (FITC-conjugated, Emfret Analytics) antibodies in parallel with RNase treatment (2 mg / mL, Invitrogen). Samples were analyzed by flow cytometer (Cytoflex, Beckman coulter) to measure % CD41+ nucleated cells (PI+ cells).

[0310] As shown in Figure 3A, mice treated with ActRIIA / B-mFc exhibited accelerated recovery of platelet counts after platelet depletion compared with vehicle-treated mice in an immune thrombocytopenic mouse model. These data suggest that ActRIIA / B-mFc may promote faster recovery from thrombocytopenia. In addition, as shown in Figures 5B-5C, CD41 expression in the bone marrow of the ActRIIA / B-mFc-treated group was significantly increased on day 10 after platelet depletion. +The number of megakaryocyte progenitors was increased by 25% and at a higher 4N ploidy level compared to the vehicle-treated group, suggesting that in acute thrombocytopenia, ActRIIA / B-mFc treatment promotes megakaryocyte differentiation, potentially by accelerating megakaryocyte precursor maturation, contributing to accelerated recovery in mice. For platelet data, statistical analysis was performed using repeated measures mixed effects modeling. Individual comparisons shown are from Tukey post-hoc tests. For CD41 data, statistical analysis was performed using one-way ANOVA and individual comparisons calculated using Tukey post-hoc tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. N=9 / group.

[0311] Example 4 - Effect of a single dose of ActRIIA / B-mFc on platelet counts Eleven-week-old C57Bl / 6 mice were given a single dose of either TBS (vehicle) or ActRIIA / B-mFc (10 mg / kg) via subcutaneous administration. Separate cohorts of mice from both dosing groups were sampled for whole blood on study days 37, 51, and 85, and platelet counts were determined using a veterinary hematology analyzer (Heska Element HT5).

[0312] As shown in Figure 4, a single dose of ActRIIA / B-mFc resulted in an increase in circulating platelets on days 37, 51, and 85. Data are expressed as mean ± standard error of the mean. Statistical analysis was performed using Student's T-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

[0313] Example 5 - Effect of ActRIIA / B-mFc on megakaryocyte precursors ex vivo Bone marrow cells from 11-week-old C57Bl / 6 mice were isolated and treated with activin A (5 mg / kg), ActRIIA / B-mFc (10 mg / kg), or a combination of both for 6 days, after which cells were harvested and analyzed using flow cytometry (N=2).

[0314] As shown in Figure 5, ex vivo Activin A treatment increased 2N ploidy levels and decreased the degree of polyploidy (reducing higher ploidy levels), indicating that Activin A was acting to prevent maturation of these cells. Ex vivo treatment with ActRIIA / B-mFc reversed the Activin-mediated changes in megakaryocyte precursors, indicating that ActRIIA / B-mFc inhibited the effect of Activin A on ploidy. Higher ploidy levels were seen in the Activin A + ActRIIA / B-mFc treated group compared to the Activin A group. Data are presented as mean ± standard error of the mean.

[0315] Example 6 - Effect of anti-activin A antibodies on platelet counts Ten-week-old C57Bl / 6 male mice were intraperitoneally administered either TBS (vehicle), anti-activin A antibody (described in WO2008031061A2, 5 mg / kg), or ActRIIA / B-mFc (10 mg / kg). Whole blood was sampled 24 hours after administration, and platelet counts were determined using a veterinary hematology analyzer (Hematrue).

[0316] As shown in Figure 6, treatment with anti-activin A antibody and ActRIIA / B-mFc increased platelets in wild-type mice. The increase in platelets observed with anti-activin A antibody suggests that inhibition of activin A may be at least partially responsible for the increased platelet levels observed with ActRIIA / B-mFc. Data are presented as mean ± standard error of the mean. Data were analyzed using one-way ANOVA with Fisher's LSD using Prism 9 (GraphPad Software, San Diego, CA, USA). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0317] Example 7 - TPO in myelofibrosis 高 Effects of ActRIIA / B-mFc on platelets, red blood cell parameters, spleen weight, and immune cells in the model TPO in myelofibrosis高 The model induces myelofibrosis-like pathology by high exposure to thrombopoietin, a natural endocrine inducer of megakaryocyte progenitor cell proliferation and development. Seven-week-old C57Bl / 6 albino mice (B6(Cg)-Tyr, Jackson Laboratory) were tail vein injected with 0.75 mg / kg of thrombopoietin (TPO) expression plasmid cloned into pLEV113 plasmid (Lake Pharma). The injection was performed with a hydrodynamic approach, where a 100 mL / kg volume was injected over a short period of time (6–10 s). On day 3 after TPO injection, mice were divided into two groups to receive either vehicle (TBS) or ActRIIA / B-mFc (7.5 mg / kg) twice weekly. Mice were sacrificed on day 14 after TPO injection. Hematological parameters were measured using a Heska Element HT5 veterinary hematology analyzer.

[0318] As shown in Figure 7, TPO HDI increased platelet count and volume. Treatment with ActRIIA / B-mFc was associated with a significant attenuation in platelet expansion. High platelet levels are associated with thrombocythemia, which may lead to secondary myelofibrosis. These data suggest that ActRIIA / B-mFc rebalances the number of cells committed to the megakaryocytic lineage. N=10-12 mice / group. Results are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. ****=p<0.0001.

[0319] Data is TPO 高 We confirmed that the myelofibrosis model became anemic 14 days after TPO overexpression (Figure 8). Treatment with ActRIIA / B-mFc was associated with significant improvements in RBC metrics and appeared to reduce the development of anemia in this model. N=10-12 mice / group. Results are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. **=p<0.01, ***=p<0.001, ****=p<0.0001.

[0320] The expanded megakaryocyte growth and proliferation reduces the bone marrow's ability to produce hematopoiesis and induces compensatory extramedullary hematopoiesis in the liver and spleen (Figure 9). These data show a significant reduction in splenomegaly in mice treated with ActRIIA / B-mFc, indicating a reduction in extramedullary hematopoiesis, likely due to a reduced need for this compensatory process. N=10-12 mice / group. Results are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. *=p<0.05, ****=p<0.0001.

[0321] Finally, as shown in Figure 10, TPO HDI led to an increase in leukocytes, neutrophils, and lymphocytes, and treatment with ActRIIA / B-mFc reduced the TPO-mediated increase in leukocytes and lymphocytes. TPO signals through the JAK2 pathway. The JAK2 pathway is proliferative and activating mutations are associated with predisposition to the development of oncological syndromes and myeloid leukemia. N=10-12 mice / group. Results are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. ***=p<0.001, ****=p<0.0001.

[0322] Example 8 - Effect of combined administration of ActRIIA / B-mFc and ruxolitinib Pharmacological evaluation was performed on mice to examine whether treatment with ActRIIA / B-mFc could overcome ruxolitinib-induced anemia. Female C57Bl / 6 mice aged 10–12 weeks were orally administered either vehicle (n=10) or ruxolitinib at 90 mg / kg (n=20) or 120 mg / kg (n=20) twice daily. After 37 days of ruxolitinib therapy, blood was sampled by buccal bleeding for hematological evaluation. On day 41, mice from each ruxolitinib dose were split into two groups to receive either TBS (vehicle) (n=10) or ActRIIA / B-mFc (7.5 mg / kg, n=10 / group) by IP twice weekly for 14 days, concurrent with the continuation of ruxolitinib dosing. Hematological parameters were assessed in whole blood using a Heska Element HT5 veterinary hematology analyzer.

[0323] As shown in FIG. 11, ActRIIA / B-mFc increased body weight in mice treated with ruxolitinib. N=10. Data are presented as the mean±standard error of the mean. Two-way ANOVA was used for statistical analysis. **=p<0.01 between vehicle and ruxolitinib 90 mg / kg+ActRIIA / B-mFc at each time point. ***=p<0.005 between vehicle and ruxolitinib 90 mg / kg+ActRIIA / B-mFc at each time point. ****=p<0.0001 between vehicle and ruxolitinib 90 mg / kg+ActRIIA / B-mFc at each time point. #=p<0.05 between vehicle and ruxolitinib 120 mg / kg+ActRIIA / B-mFc at each time point. ## = p<0.01 between vehicle and ruxolitinib 120 mg / kg + ActRIIA / B-mFc at each time point. This demonstrates that ruxolitinib + ActRIIA / B-mFc combination therapy did not adversely affect body weight, a common marker of tolerability. Furthermore, the ability to increase body weight may be of potential benefit in elderly myelofibrosis patients.

[0324] As shown in Figure 12, treatment with ruxolitinib alone reduced RBC volume, hemoglobin, and hematocrit. Data are presented as mean ± standard error of the mean. Vehicle N=10, ruxolitinib 90 N=20, ruxolitinib 120 N=20. One-way ANOVA followed by Dunnett's post-hoc test was used for statistical analysis. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. However, administration of ActRIIA / B-mFc abolished the ruxolitinib-associated reduction in RBC volume, hemoglobin, and hematocrit (Figure 13). This indicates that ActRIIA / B-mFc functions independently of the JAK / STAT pathway, suggesting that ActRIIA / B-Fc may be a potential therapeutic option for ineffective hematopoiesis caused by defective JAK / STAT signaling in myelofibrosis patients. Treatment with ActRIIA / B-Fc may alleviate the dose-limiting effect of ruxolitinib and extend the duration of therapy in myelofibrosis patients. Data are expressed as mean ± standard error of the mean. N=10 for all groups. One-way ANOVA followed by Tukey post-hoc test was used for statistical analysis. ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

[0325] Example 9 - Megakaryocyte precursors express activin, GDF, BMP, and TGF-β ligands and their cognate receptors. Bone marrow from three naive mice was pooled and selected for the megakaryocyte marker CD41. After cells underwent positive selection using rat anti-mouse CD41 (clone-MWReg30), RNA was extracted using Zymo Research Direct-zol RNA MicroPrep Kit. Total RNA was then converted to cDNA (QuantiTect Reverse Transcription Kit) and at least 20 ng per well was added to a mouse-specific TGF-β family pathway TaqMan gene expression array (ThermoFisher Custom Gene Array). Quantitative real-time PCR was performed and results were plotted using 2^delta Ct values. TGF-β gene expression was normalized using housekeeping genes including 18s, Gusb, Gapdh, Actb, and Ubc. Results are shown in Figures 14A-14B and represent three independent experimental replicates. As shown in Figures 14A-14B, mouse bone marrow megakaryocyte precursors expressed activin, GDF, BMP, and TGF-β ligands and their cognate receptors, including activin receptors IIA and IIB. These data demonstrate the ability of the TGF-β pathway to participate in megakaryocyte differentiation and normal megakaryocyte function. The gene encoding the activin A protein, INHBA, was moderately expressed compared to the ligands of other family members. Receptors and ligands directly related to ActRIIA / B-mFc are bolded. ND = not detected.

[0326] Example 10 - Treatment of anemia in subjects receiving ruxolitinib for myelofibrosis by co-administration of an ActRIIA ligand trap containing an extracellular ActRIIA variant According to the methods disclosed herein, one skilled in the art can treat a subject, e.g., a human patient, who has received ruxolitinib for the treatment of myelofibrosis (e.g., PMF, post-ET MF, and post-PV MF) and has anemia to increase red blood cell count, increase hemoglobin levels, increase hematocrit, reduce RBC transfusions, promote transfusion independence, and / or treat anemia. The treatment method can include diagnosing or identifying the subject as a treatment candidate by measuring hemoglobin levels. To treat the subject, one skilled in the art can administer to the subject a composition comprising an ActRIIA ligand trap comprising an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). The composition comprising an ActRIIA ligand trap comprising an extracellular ActRIIA variant may be administered to the subject, for example, by parenteral injection (e.g., intravenous or subcutaneous injection) in combination with ruxolitinib orally administered once or twice a day. An ActRIIA ligand trap containing an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) is administered in a therapeutically effective amount, for example, 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.325, 0.35, 0.375, , 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIA variant is administered every other month, 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 or more times per week).An ActRIIA ligand trap comprising an extracellular ActRIIA variant is administered in an amount sufficient to increase red blood cell count, increase hemoglobin levels, increase hematocrit, reduce RBC transfusions, promote transfusion independence, and / or treat anemia.

[0327] After administering the composition to a patient, a physician skilled in the art can monitor the improvement of the patient in response to therapy by various methods.For example, the physician can use blood tests to monitor the patient's red blood cell count, hemoglobin level, or hematocrit.The observation that the patient's red blood cell count, hemoglobin level, or hematocrit increases after administering the composition compared to the test results before administering the composition indicates that the patient is responding favorably to treatment.Subsequent doses can be determined and administered as necessary.

[0328] Other embodiments While the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and that this application is generally intended to cover any changes, uses, or adaptations of the invention which follow the principles of the invention and are within known or customary practice within the art to which the invention pertains, including departures from the invention which may be applied to the essential features described above, and are within the scope of the appended claims. Other embodiments are within the scope of the claims.

Claims

1. A medicament for treating cytopenia in a subject, the medicament comprising a combination of an ActRII signaling inhibitor and a JAK inhibitor and administered to the subject; The ActRII signaling inhibitor is a polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant, wherein the variant has the amino acid sequence of SEQ ID NO:

69.

2. The pharmaceutical described in claim 1, wherein the subject has myelofibrosis.

3. The pharmaceutical composition of claim 2, wherein the myelofibrosis is intermediate-risk or high-risk myelofibrosis.

4. The pharmaceutical composition of claim 2, wherein the myelofibrosis is primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (post-ET MF), or post-polycythemia vera myelofibrosis (post-PV MF).

5. A pharmaceutical described in any one of claims 1 to 4, wherein the cytopenia is anemia, thrombocytopenia, or neutropenia.

6. The pharmaceutical composition according to claim 5, wherein the cytopenia is anemia.

7. A pharmaceutical described in any one of claims 1 to 4, wherein the JAK inhibitor is ruxolitinib, fedratinib, or pacritinib.

8. The pharmaceutical described in claim 7, wherein the JAK inhibitor is ruxolitinib.

9. A pharmaceutical described in any one of claims 1 to 4, wherein the polypeptide further comprises an Fc domain.

10. The pharmaceutical described in claim 9, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

79.

11. The pharmaceutical described in claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

80.

12. A pharmaceutical described in any one of claims 1 to 4, wherein the subject is transfusion dependent.

13. The pharmaceutical described in claim 12, wherein the subject has a low transfusion burden.

14. The pharmaceutical described in claim 12, wherein the subject has a high transfusion burden.

15. A pharmaceutical described in any one of claims 1 to 4, wherein the subject has been treated with a JAK inhibitor or is currently being treated with a JAK inhibitor.