Treatment of beta-thalassemia using actrii ligand trap

Abstract

This article relates to the treatment of β-thalassemia using ACTRII ligand traps. This article provides a method for treating β-thalassemia by subcutaneous administration of an ActRII signaling inhibitor at approximately 0.8 mg / kg. This article also provides a method for adjusting the dose of the ActRII signaling inhibitor administered to the recipient.

Description

[0001] 1. Cross-references to related applications This application is a divisional application of Chinese Patent Application No. 201680041002.2, filed on January 11, 2018, entitled "Treatment of β-thalassemia using ACTRII ligand trap".

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 161,136, filed May 13, 2015; U.S. Provisional Patent Application No. 62 / 173,836, filed June 10, 2015; and U.S. Provisional Patent Application No. 62 / 243,457, filed October 19, 2015, the entire contents of each of which are incorporated herein by reference and used for all purposes.

[0003] 2. Sequence List This application is being submitted along with a sequence list with the filename "12827_952_228_SeqListing.txt", which is 97 kilobytes in size and was created on May 4, 2016. The sequence list is incorporated herein by reference in its entirety and for all purposes. Invention Field This article provides methods for treating and / or preventing β-thalassemia, such as transfusion-dependent and non-transfusion-dependent β-thalassemia, the methods comprising administering a target activin type II receptor signaling inhibitor (ActRII signaling inhibitor, such as an activin ligand trap). Background of the Invention Beta-thalassemia, one of the most common inherited hemoglobinopathies worldwide, is caused by an autosomal mutation in the gene encoding β-globin, which induces the absence or low-level synthesis of this protein in erythropoietic cells (Weatherall DJ, 2001, Nature Reviews Genetics; 2(4):245-255). Approximately 80-90 million people (about 1.5% of the global population) are carriers of beta-thalassemia, with about 60,000 symptomatic individuals born each year (Modell et al., 2007, Scand J Clin Lab Invest; 67:39-69). The annual incidence rate in symptomatic individuals is estimated at 1 / 100,000 worldwide and 1 / 10,000 in the European Union (EU) (Galanello R and Origa R, 2010, Orphanet J Rare Dis; 5:11). The incidence rate is highest in the Mediterranean region, the Middle East and Southeast Asia (particularly India, China and Indonesia; this region accounts for about 50% of affected births), and the incidence rate is gradually increasing worldwide due to migration (e.g. Europe, the United States and Australia) (Colah R, Gorakshakar et al., 2010; Expert Rev Hematol; 3(1):103-17; Modell et al., 2008, Bull World Health Organ; 86(6):480-7).

[0004] β-thalassemia is characterized by a reduction in β-globin chains, followed by an imbalance in the globin chain (α:non-α ratio) of the hemoglobin (Hb) molecule, leading to impaired erythropoiesis and other complications. Nearly 200 different mutations affecting the β-globin gene have been described in patients with β-thalassemia, and these mutations can be homozygous or complex heterozygous. Therefore, phenotypic effects vary considerably from mild impairment to complete inhibition of β-globin chain synthesis (Thein SL, 2013, Cold Spring Harb Perspect Med;3(5):a011700). In addition to defective β-globin chains, patients may also have β-thalassemia combined with structural variants (e.g., HbE), resulting in HbE / β-thalassemia.

[0005] Given the current lack of safe and effective drug therapies for treating β-thalassemia (e.g., transfusion-dependent and non-transfusion-dependent β-thalassemia), there is a significant unmet medical need for novel therapies specifically addressing the underlying pathophysiology of β-thalassemia syndromes, including anemia and complications of ineffective erythropoiesis.

[0006] Two related type II receptors, ActRIIA and ActRIIB, have been identified as type II receptors for activin (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins, ActRIIA and ActRIIB can biochemically interact with several other TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Leeand McPherron, 2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is a type I receptor for activins, particularly activin A, while ALK-7 can also act as a receptor for activins, particularly activin B.

[0007] An activin ligand trap, consisting of a humanized fusion protein including the extracellular domain of activin receptor type IIB (ActRIIB) and human IgG1 Fc (ActRIIB-hFc), is currently being evaluated in a phase II clinical trial for the treatment of subjects with β-thalassemia. Invention Overview This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject at a dose of about 0.8 mg / kg or about 1.0 mg / kg, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject in the upper arm, abdomen or thigh every 21 days.

[0008] This article provides a method for treating transfusion-dependent β-thalassemia in subjects in need, the method comprising administering an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen or thigh every 21 days.

[0009] This article provides a method for treating transfusion-independent β-thalassemia in subjects in need, the method comprising administering an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject in the upper arm, abdomen or thigh every 21 days.

[0010] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject at approximately 0.8 mg / kg or approximately 1.0 mg / kg, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, wherein the subject's phenotype is selected from β-thalassemia. 0 / β 0 β + / β + β 0 / β + β 0 / HbE and β + / HbE.

[0011] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject at approximately 0.8 mg / kg or approximately 1.0 mg / kg, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject in the upper arm, abdomen or thigh every 21 days, wherein the subject's phenotype includes co-inherited two severe hemoglobin β-chain mutations, and wherein the subject has α-thalassemia.

[0012] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject at approximately 0.8 mg / kg or approximately 1.0 mg / kg, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously in the subject's upper arm, abdomen, or thigh, wherein the subject's phenotype includes co-inherited two severe hemoglobin β-chain mutations, and wherein the subject suffers from hereditary fetal hemoglobin persistence.

[0013] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject at approximately 0.8 mg / kg or approximately 1.0 mg / kg, followed by administering the ActRII signaling inhibitor to the subject once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh.

[0014] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject, followed by administration of the ActRII signaling inhibitor once or multiple times at 21-day intervals, thereby treating β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the subject's phenotype is selected from β-thalassemia. 0 / β 0 β + / β + β 0 / β + β 0 / HbE and β + / HbE.

[0015] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, followed by administration of the ActRII signaling inhibitor once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the subject suffers from hereditary fetal hemoglobin persistence.

[0016] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type II (ActRII) signaling inhibitor to the subject, followed by administering the ActRII signaling inhibitor to the subject once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the administration is sufficient to detectably reduce the subject's serum GDF-11 level between administrations.

[0017] In some embodiments of any of the foregoing methods, β-thalassemia is transfusion-dependent β-thalassemia. In some embodiments of any of the foregoing methods, β-thalassemia is non-transfusion-dependent β-thalassemia.

[0018] In some embodiments of any of the foregoing methods, the method further includes performing a first measurement of the subject's hemoglobin concentration; performing a second measurement of the subject's hemoglobin concentration after a first period of time; and administering a subsequent dose of an ActRII signaling inhibitor based on the difference between the second measurement of hemoglobin concentration and the first measurement of hemoglobin concentration, wherein the administration includes subcutaneous administration to the subject's upper arm, abdomen, or thigh.

[0019] In some embodiments of any of the foregoing methods, the method further includes performing a first measurement of the subject's hematocrit; performing a second measurement of the subject's hematocrit after a first period of time; and administering a subsequent dose of an ActRII signal transduction inhibitor based on the difference between the second measurement of hematocrit and the first measurement of hematocrit, wherein the administration includes subcutaneous administration to the subject's upper arm, abdomen, or thigh.

[0020] In some embodiments of any of the foregoing methods, the method further includes performing a first measurement of the subject's fetal hemoglobin; performing a second measurement of the subject's fetal hemoglobin concentration after a first period of time; and administering a subsequent dose of an ActRII signal transduction inhibitor based on the difference between the second measurement of fetal hemoglobin concentration and the first measurement of fetal hemoglobin concentration, wherein the administration includes subcutaneous administration in the subject's upper arm, abdomen, or thigh.

[0021] In some embodiments of any of the foregoing methods, the method further includes (a) performing a first measurement of the subject's hemoglobin concentration, hematocrit, or fetal hemoglobin concentration; (b) performing a second measurement of the subject's hemoglobin concentration, hematocrit, or fetal hemoglobin concentration after a first time period; and (c) after a second time period, terminating the administration of the initial dose and administering a subsequent dose of an ActRII signaling inhibitor to the subject, wherein the subsequent dose is administered subcutaneously in the subject's upper arm, abdomen, or thigh.

[0022] In some embodiments of any of the foregoing methods, the first measurement of hemoglobin concentration, hematocrit, or fetal hemoglobin concentration is performed before administering the initial dose of the ActRII signaling inhibitor to the subject. In some embodiments, the first measurement of hemoglobin concentration, hematocrit, or fetal hemoglobin concentration is performed immediately after administering the initial dose of the ActRII signaling inhibitor to the subject, or within up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week thereafter. In some embodiments, a second measurement of hemoglobin, hematocrit, or fetal hemoglobin concentration is performed approximately 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after administering the initial dose of the ActRII signaling inhibitor to the subject. In some embodiments, the second time period is within 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks after the second measurement. In some embodiments, the subsequent dose of the ActRII signaling inhibitor is about 0.3 mg / kg, about 0.45 mg / kg, about 0.6 mg / kg, about 1.0 mg / kg, or about 1.25 mg / kg. In some embodiments, the method further includes performing a third measurement of the subject's hemoglobin concentration, hematocrit, or fetal hemoglobin concentration.

[0023] In some embodiments of any of the foregoing methods, (a) the second measurement of hemoglobin concentration is less than or equal to 12.5 g / dL; (b) the second measurement of hemoglobin concentration is greater than the first measurement of hemoglobin concentration which is less than or equal to 1.5 g / dL; and (c) the subsequent dose is equal to the initial dose.

[0024] In some embodiments of any of the foregoing methods, (a) the second measurement of hemoglobin concentration is less than or equal to 12.5 g / dL; (b) the second measurement of hemoglobin concentration is greater than the first measurement of hemoglobin concentration by more than 1.5 g / dL; and (c) the subsequent dose is less than about 25% of the initial dose.

[0025] In some embodiments of any of the foregoing methods, (a) a second measurement of hemoglobin concentration (i) is greater than 12.5 g / dL and less than or equal to 14 g / dL; and (ii) is greater than the first measurement of hemoglobin concentration but less than or equal to 1.5 g / dL; (b) subsequent doses are equal to the initial dose; and (c) a second period includes a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is less than or equal to 12.5 g / dL.

[0026] In some embodiments of any of the foregoing methods, (a) a second measurement of hemoglobin concentration (i) is greater than 12.5 g / dL and less than or equal to 14 g / dL, and (ii) is greater than the first measurement of hemoglobin concentration by more than 1.5 g / dL; (b) a subsequent dose is less than about 25% of the initial dose; and (c) a second period comprising a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is determined to be (i) less than or equal to 12.5 g / dL, and (ii) the change between the first and third measurements of hemoglobin concentration is less than or equal to 1.5 g / dL.

[0027] In some embodiments of any of the foregoing methods, (a) a second measurement of hemoglobin concentration is greater than 14 g / dL; (b) a subsequent dose is less than about 25% of the initial dose; and (c) a second period includes a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is less than 12.5 g / dL.

[0028] In some embodiments of any of the foregoing methods, the initial dose is administered every 21 days. In some embodiments, subsequent doses are administered every 21 days.

[0029] In some embodiments of any of the foregoing methods, the method further includes reducing the subject's GDF11 level. In some embodiments of any of the foregoing methods, the method further includes increasing the subject's fetal hemoglobin level.

[0030] In some embodiments of any of the foregoing methods, the ActRII signaling inhibitor is an inhibitor of ActRIIA signaling. In some embodiments, the ActRII signaling inhibitor is a humanized fusion protein composed of the extracellular domain of ActRIIA and the human IgG1 Fc domain. In some embodiments, the ActRIIA signal transduction inhibitor is a polypeptide comprising a sequence of amino acids selected from the following: (a) 90% identity with SEQ ID NO:2; (b) 95% identity with SEQ ID NO:2; (c) 98% identity with SEQ ID NO:2; (d) SEQ ID NO:2; (e) 90% identity with SEQ ID NO:3; (f) 95% identity with SEQ ID NO:3; (g) 98% identity with SEQ ID NO:3; (h) SEQ ID NO:3; (i) 90% identity with SEQ ID NO:6; (j) 95% identity with SEQ ID NO:6; (k) 98% identity with SEQ ID NO:6; (l) SEQ ID NO:6; (m) 90% identity with SEQ ID NO:7; (n) 95% identity with SEQ ID NO:7; (o) SEQ ID NO:2; (i) 90% identity with SEQ ID NO:7; (ii) 90% identity with SEQ ID NO:7; (iii) 90% identity with SEQ ID NO:7; (iv) 90% identity with SEQ ID NO:6; (v) 90% identity with SEQ ID NO:7; (v ...6; (v) 90% identity with SEQ ID NO:6; (v) 90% identity with SEQ ID NO NO:7 has 98% identity; and (p)SEQ ID NO:7. In some embodiments, the ActRII signal transduction inhibitor is a polypeptide containing the amino acid sequence of SEQ ID NO:7.

[0031] In some embodiments of any of the foregoing methods, the ActRII signaling inhibitor is an inhibitor of ActRIIB signaling. In some embodiments, the ActRII signaling inhibitor is a humanized fusion protein composed of the extracellular domain of ActRIIB and the human IgG1 Fc domain. In some embodiments, the ActRIIB inhibitor is a polypeptide comprising a sequence of amino acids selected from the following: (a) 90% identity with SEQ ID NO:17; (b) 95% identity with SEQ ID NO:17; (c) 98% identity with SEQ ID NO:17; (d) SEQ ID NO:17; (e) 90% identity with SEQ ID NO:20; (f) 95% identity with SEQ ID NO:20; (g) 98% identity with SEQ ID NO:20; (h) SEQ ID NO:20; (i) 90% identity with SEQ ID NO:21; (j) 95% identity with SEQ ID NO:21; (k) 98% identity with SEQ ID NO:21; (l) SEQ ID NO:21; (m) 90% identity with SEQ ID NO:25; (n) 95% identity with SEQ ID NO:25; (o) and SEQ ID NO:25. NO:25 has 98% identity; and (p) SEQ ID NO:25. In some embodiments, the ActRIIB signal transduction inhibitor is a polypeptide containing the amino acid sequence of SEQ ID NO:25.

[0032] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of approximately 0.8 mg / kg of an activin receptor type IIB (ActRIIB) signal transduction inhibitor to the subject, wherein the activin receptor type IIB (ActRIIB) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, and wherein the ActRIIB signal transduction inhibitor comprises the amino acid sequence of SEQ ID NO:25.

[0033] This article provides a method for treating transfusion-dependent β-thalassemia in subjects of need, the method comprising administering an initial dose of approximately 0.8 mg / kg of an activin receptor type IIB (ActRIIB) signal transduction inhibitor to the subject, wherein the activin receptor type II (ActRIIB) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, and wherein the ActRIIB signal transduction inhibitor comprises the amino acid sequence of SEQ ID NO:25.

[0034] This article provides a method for treating transfusion-independent β-thalassemia in subjects of need, the method comprising administering an initial dose of approximately 0.8 mg / kg of an activin receptor type IIB (ActRIIB) signal transduction inhibitor to the subject, wherein the activin receptor type IIB (ActRIIB) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, and wherein the ActRIIB signal transduction inhibitor comprises the amino acid sequence of SEQ ID NO:25.

[0035] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of approximately 0.8 mg / kg of an activin receptor type IIB (ActRIIB) signaling inhibitor to the subject, wherein the activin receptor type IIB (ActRIIB) signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, and wherein the subject's phenotype is selected from β-thalassemia. 0 / β 0 β + / β + β 0 / β + β 0 / HbE and β + / HbE, wherein the ActRIIB signal transduction inhibitor contains the amino acid sequence of SEQ ID NO:25.

[0036] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type IIB (ActRIIB) signal transduction inhibitor to the subject at a dose of about 0.8 mg / kg or about 1.0 mg / kg, wherein the activin receptor type IIB (ActRIIB) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, wherein the subject's phenotype includes co-inherited two severe hemoglobin β-chain mutations, wherein the subject has α-thalassemia, and wherein the ActRIIB signal transduction inhibitor comprises the amino acid sequence of SEQ ID NO:25.

[0037] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type IIB (ActRIIB) signal transduction inhibitor to the subject, wherein the activin receptor type IIB (ActRIIB) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, wherein the subject's phenotype includes co-inherited two severe hemoglobin β-chain mutations, wherein the subject suffers from hereditary fetal hemoglobin persistence, and wherein the ActRIIB signal transduction inhibitor comprises the amino acid sequence of SEQ ID NO:25.

[0038] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type IIB (ActRIIB) signaling inhibitor to the subject, followed by administering the ActRIIB signaling inhibitor to the subject once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh.

[0039] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type IIB (ActRIIB) signaling inhibitor to the subject, followed by administering the ActRIIB signaling inhibitor to the subject once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the subject's phenotype is selected from β-thalassemia. 0 / β 0 β + / β + β 0 / β + β 0 / HbE and β + / HbE.

[0040] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type IIB (ActRIIB) signaling inhibitor to the subject at approximately 0.8 mg / kg or approximately 1.0 mg / kg, followed by administration of the ActRIIB signaling inhibitor once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the subject suffers from hereditary fetal hemoglobin persistence.

[0041] This article provides a method for treating β-thalassemia in subjects of need, the method comprising administering an initial dose of an activin receptor type IIB (ActRIIB) signaling inhibitor to the subject, followed by administering the ActRIIB signaling inhibitor to the subject once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the administration is sufficient to detectably reduce the subject's serum GDF-11 level between administrations.

[0042] In some embodiments of any of the foregoing methods, β-thalassemia is transfusion-dependent β-thalassemia. In some embodiments of any of the foregoing methods, β-thalassemia is non-transfusion-dependent β-thalassemia.

[0043] In some embodiments of any of the foregoing methods, the method further includes performing a first measurement of the subject's hemoglobin concentration; performing a second measurement of the subject's hemoglobin concentration after a first period of time; and administering a subsequent dose of an ActRIIB signal transduction inhibitor based on the difference between the second measurement of hemoglobin concentration and the first measurement of hemoglobin concentration, wherein the administration includes subcutaneous administration to the subject's upper arm, abdomen, or thigh.

[0044] In some embodiments of any of the foregoing methods, the method further includes performing a first measurement of the subject's hematocrit; performing a second measurement of the subject's hematocrit after a first period of time; and administering a subsequent dose of an ActRIIB signal transduction inhibitor based on the difference between the second measurement of hematocrit and the first measurement of hematocrit, wherein the administration includes subcutaneous administration to the subject's upper arm, abdomen, or thigh.

[0045] In some embodiments of any of the foregoing methods, the method further includes performing a first measurement of the subject's fetal hemoglobin concentration; performing a second measurement of the subject's fetal hemoglobin concentration after a first period of time; and administering a subsequent dose of an ActRIIB signal transduction inhibitor based on the difference between the second measurement of fetal hemoglobin concentration and the first measurement of fetal hemoglobin concentration, wherein the administration includes subcutaneous administration in the subject's upper arm, abdomen, or thigh.

[0046] In some embodiments of any of the foregoing methods, the method further includes (a) performing a first measurement of the subject's hemoglobin concentration; (b) performing a second measurement of the subject's hemoglobin concentration after a first period of time; and (c) after a second period of time, terminating the administration of the initial dose and administering a subsequent dose of an ActRIIB signaling inhibitor to the subject, wherein the subsequent dose is administered by subcutaneous injection into the subject's upper arm, abdomen, or thigh.

[0047] In some embodiments of any of the foregoing methods, the first measurement of hemoglobin concentration, hematocrit, or fetal hemoglobin concentration is performed before administering the initial dose of the ActRIIB signaling inhibitor to the subject. In some embodiments, the first measurement of hemoglobin concentration, hematocrit, or fetal hemoglobin concentration is performed immediately after administering the initial dose of the ActRIIB signaling inhibitor to the subject, or at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week thereafter. In some embodiments, a second measurement of hemoglobin concentration, hematocrit, or fetal hemoglobin concentration is performed approximately 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after administering the initial dose of the ActRIIB signaling inhibitor to the subject. In some embodiments, the second time period is within 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks after the second measurement. In some embodiments, the subsequent dose of the ActRIIB signaling inhibitor is about 0.3 mg / kg, about 0.45 mg / kg, about 0.6 mg / kg, about 1.0 mg / kg, or about 1.25 mg / kg. In some embodiments, the method further includes performing a third measurement of the subject's hemoglobin concentration, hematocrit, or fetal hemoglobin concentration.

[0048] In some embodiments of any of the foregoing methods, (a) the second measurement of hemoglobin concentration is less than or equal to 12.5 g / dL; (b) the second measurement of hemoglobin concentration is greater than the first measurement of hemoglobin concentration which is less than or equal to 1.5 g / dL; and (c) the subsequent dose is equal to the initial dose.

[0049] In some embodiments of any of the foregoing methods, (a) the second measurement of hemoglobin concentration is less than or equal to 12.5 g / dL; (b) the second measurement of hemoglobin concentration is greater than the first measurement of hemoglobin concentration by more than 1.5 g / dL; and (c) the subsequent dose is less than about 25% of the initial dose.

[0050] In some embodiments of any of the foregoing methods, (a) a second measurement of hemoglobin concentration (i) is greater than 12.5 g / dL and less than or equal to 14 g / dL; and (ii) is greater than the first measurement of hemoglobin concentration but less than or equal to 1.5 g / dL; (b) subsequent doses are equal to the initial dose; and (c) a second period includes a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is less than or equal to 12.5 g / dL.

[0051] In some embodiments of any of the foregoing methods, (a) a second measurement of hemoglobin concentration (i) is greater than 12.5 g / dL and less than or equal to 14 g / dL, and (ii) is greater than the first measurement of hemoglobin concentration by more than 1.5 g / dL; (b) a subsequent dose is less than about 25% of the initial dose; and (c) a second period comprising a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is determined to be (i) less than or equal to 12.5 g / dL, and (ii) the change between the first and third measurements of hemoglobin concentration is less than or equal to 1.5 g / dL.

[0052] In some embodiments of any of the foregoing methods, (a) a second measurement of hemoglobin concentration is greater than 14 g / dL; (b) a subsequent dose is less than about 25% of the initial dose; and (c) a second period includes a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is less than 12.5 g / dL.

[0053] In some embodiments of any of the foregoing methods, the initial dose is administered every 21 days. In some embodiments of any of the foregoing methods, subsequent doses are administered every 21 days.

[0054] In some embodiments of any of the foregoing methods, the method further includes reducing the GDF11 level of the object.

[0055] In some embodiments of any of the foregoing methods, the method further includes increasing the fetal hemoglobin level of the subject.

[0056] This article provides a method for increasing fetal hemoglobin levels in a subject, the method comprising administering an ActRIIB signaling inhibitor to the subject.

[0057] In some implementations of any of the aforementioned methods, the object expresses hemoglobin E.

[0058] In some implementations of any of the aforementioned methods, the object does not express hemoglobin S.

[0059] In some embodiments of any of the foregoing methods, the erythroid response consists of: (i) a reduction in transfusion burden of 33% or more for 12 weeks, and (ii) a reduction of at least 2 units of red blood cells over the 12-week period.

[0060] In some embodiments of any of the foregoing methods, the red blood cell response includes an increase in hemoglobin concentration greater than 1 g / dL compared to baseline hemoglobin concentration, wherein the increase in hemoglobin concentration is measured by the hemoglobin concentration value during a continuous 12-week period without transfusion.

[0061] In some implementations of any of the aforementioned methods, the object is a person.

[0062] In some embodiments of any of the foregoing methods, the ActRII signaling inhibitor is packaged in a sterile, preservative-free lyophilized block and stored between 2°C and 8°C before administration. In some embodiments, the container contains 37.5 mg of the ActRII signaling inhibitor. In some embodiments, the container contains 75 mg of the ActRII signaling inhibitor. Brief description of the attached diagram Figure 1 This describes the healing of leg ulcers in exemplary transfusion-dependent patients before treatment or after receiving ActRIIB-hFc (SEQ ID NO:25) at a dose of 1.25 mg / kg for 2 or 5 weeks. Invention Details 7.1 Overview This article provides a method for treating subjects with β-thalassemia (e.g., transfusion-dependent or non-transfusion-dependent β-thalassemia), the method comprising administering an ActRII signaling inhibitor to the subject.

[0063] 7.2 Abbreviations and Terms As used herein, the term "about" when used with a numerical value refers to any value within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the referenced value. In some embodiments, the term "about" includes the exact numerical value listed.

[0064] As used herein, “ActRII” refers to activin receptor type II. As used herein, “ActRIIA” refers to activin receptor type IIA. See, for example, Mathews and Vale, 1991, Cell 65:973-982. GenBank™ accession number NM_001278579.1 provides an exemplary human ActRIIA nucleic acid sequence. GenBank™ accession number NP_001265508.1 provides an exemplary human ActRIIA amino acid sequence. As used herein, “ActRIIB” refers to activin receptor type IIB. See, for example, Attisano et al., 1992, Cell 68:97-108. GenBank™ accession number NM_001106.3 provides an exemplary human ActRIIB nucleic acid sequence. GenBank™ accession number NP_001097.2 provides an exemplary human ActRIIB amino acid sequence.

[0065] As used herein, “ActRIIA-mFc” or “mActRIIA-Fc” refers to a mouse activin IIA receptor-IgG1 fusion protein. See, for example, U.S. Patent No. 8,173,601. As used herein, “mActRIIB-Fc” or “ActRIIB-mFc” refers to a mouse activin IIA receptor-IgG1 fusion protein. See, for example, U.S. Patent No. 8,173,601. As used herein, “hActRIIA-Fc” or “ActRIIA-hFc” refers to a human activin IIA receptor-IgG1 fusion protein. See, for example, U.S. Patent No. 8,173,601. In some embodiments, ActRIIA-hFc refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:7. As used herein, “hActRIIB-Fc” or “ActRIIB-hFc” refers to a human activin IIA receptor-IgG1 fusion protein. See, for example, U.S. Patent No. 8,173,601. In some implementations, ActRIIB-hFc refers to a polypeptide containing the amino acid sequence of SEQ ID NO:25.

[0066] “AE” refers to an adverse event.

[0067] “β 0 "" refers to alleles associated with the absence of β-globin subunit synthesis.

[0068] “β + "" refers to alleles associated with reduced synthesis of β-globin subunits.

[0069] “Hb” refers to hemoglobin protein. GenBank™ accession number NP_000549.1 (SEQ ID NO:48) provides an exemplary amino acid sequence of the human hemoglobin α subunit. GenBank™ accession number NP_000509.1 (SEQ ID NO:49) provides an exemplary amino acid sequence of the human hemoglobin β subunit. GenBank™ accession number NP_000550.2 (SEQ ID NO:50) provides an exemplary amino acid sequence of the human hemoglobin γ subunit. Typically, the most common form of hemoglobin in adults contains two α subunits and two β subunits. Fetal hemoglobin, also known as “hemoglobin F” or “HbF”, contains two α subunits and two γ subunits.

[0070] "HbE" or "hemoglobin E" is a technically accepted term referring to a mutated form of hemoglobin (such as human hemoglobin). Hemoglobin E contains two α subunits and two β subunits, with the β subunit having a glutamic acid residue at position 26 replaced by a lysine residue (E26K).

[0071] "HbE / β-thalassemia" refers to anemia where hemoglobin E and β-thalassemia are both high and low in hemoglobin.0 Co-inheritance of alleles.

[0072] "HbS" or "hemoglobin S" is a technically accepted term referring to a mutated form of hemoglobin (such as human hemoglobin). Hemoglobin S contains two α subunits and two β subunits, with the β subunit having a glutamine-valine mutation at position 6 (G6V).

[0073] In some implementations, one unit of red blood cells refers to the amount of concentrated red blood cells obtained from approximately 400-500 mL of donated blood.

[0074] 7.3 Treatment and / or prevention methods 7.3.1 β-Thalassemia In some embodiments, this document provides a method for treating and / or preventing β-thalassemia in a subject, the method comprising administering to the subject an initial dose of about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, or about 1.1 mg / kg of an ActRII signaling inhibitor (e.g., an activin ligand trap), wherein the ActRII signaling inhibitor is administered subcutaneously to the subject in the upper arm, abdomen, or thigh.

[0075] In some embodiments, this document provides a method for treating and / or preventing β-thalassemia in a subject, the method comprising administering an initial dose of approximately 0.8 mg / kg of an ActRII signaling inhibitor (e.g., an activin ligand trap) to the subject, wherein the ActRII signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh.

[0076] In some implementations, “treatment,” “cure,” or “management” in the case of β-thalassemia includes improving at least one symptom of β-thalassemia. Non-limiting examples of symptoms of β-thalassemia include defective erythrocyte production in the bone marrow, ineffective erythropoiesis, insufficient hemoglobin levels, multiple organ dysfunction, iron overload, pallor, fatigue, jaundice, and splenomegaly.

[0077] In some embodiments, the object is the object described in Section 7.5. In some embodiments, β-thalassemia is transfusion-dependent β-thalassemia. In some embodiments, β-thalassemia is non-transfusion-dependent β-thalassemia.

[0078] In some embodiments, the ActRII signal transduction inhibitor is as described in Section 5.6. In some embodiments, the ActRII signal transduction inhibitor is the ActRIIB signal transduction inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signal transduction inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25). In some embodiments, the ActRII signal transduction inhibitor is an ActRIIA signal transduction inhibitor as described in Section 7.6.1. In some embodiments, the ActRIIA signal transduction inhibitor is an ActRIIA-Fc, such as ActRIIA-hFc (e.g., SEQ ID NO:7).

[0079] In some implementations, the ActRII signaling inhibitor is administered to the subject as described in Section 7.9.

[0080] In some implementations, the ActRII signaling inhibitor is administered to the subject together with a second drug active agent or therapy, as described in Section 7.8.

[0081] In some embodiments, the method further includes administering a subsequent dose of the ActRII signaling inhibitor to the subject as described in Section 7.3.2 or Section 7.4. For example, the method may further include analysis of the subject's hemoglobin concentration as a means of determining the subsequent dosing regimen for the subject. In some embodiments, the subject's hemoglobin concentration may be used to (i) evaluate the appropriateness of dosing for the subject, wherein the subject is a candidate for or currently receiving ActRII signaling inhibitor treatment (e.g., activin ligand trap); (ii) evaluate whether the dose of the ActRII signaling inhibitor has been adjusted during treatment; and / or (iii) evaluate the appropriate maintenance dose of the ActRII signaling inhibitor. Dosing with the ActRII signaling inhibitor may be initiated, increased, decreased, delayed, or terminated based on the subject's hemoglobin concentration. See, for example, Tables 1 and 2. In some embodiments, the method further includes (a) performing a first measurement of the subject's hemoglobin concentration; (b) performing a second measurement of the subject's hemoglobin concentration after a first time period; and (c) after a second time period, terminating the initial dose and administering a subsequent dose of an ActRII signaling inhibitor to the subject, wherein the subsequent dose is administered subcutaneously in the subject's upper arm, abdomen, or thigh. In some embodiments, the method further includes performing a third measurement of the subject's hemoglobin concentration. In some embodiments, the subsequent dose of the ActRII signaling inhibitor is titrated up to a maximum subsequent dose of approximately 1.25 mg / kg. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRIIB signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0082] In some embodiments, treating a subject according to the methods provided herein (e.g., the subject described in Section 7.5) induces a red blood cell response in the subject. In some embodiments, the red blood cell response includes a reduction in the subject's transfusion burden of at least 33%, wherein the subject has transfusion-dependent β-thalassemia. In some embodiments, the red blood cell response includes a reduction in the subject's transfusion burden of at least 50%, wherein the subject has transfusion-dependent β-thalassemia. In some embodiments, the red blood cell response includes a reduction in the subject's transfusion burden of at least 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%, wherein the subject has transfusion-dependent β-thalassemia. In some embodiments, a red blood cell response includes a reduction in the transfusion burden of a subject of at least 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% for at least 8, 9, 10, 11, 12, 13, 14, 15, 16 weeks, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, wherein the subject has transfusion-dependent β-thalassemia. In some embodiments, a red blood cell response includes a reduction in the transfusion burden of a subject of at least 33% for at least 12 weeks, wherein the subject has transfusion-dependent β-thalassemia. In some embodiments, a red blood cell response includes a reduction in the transfusion burden of a subject of at least 50% for at least 12 weeks, wherein the subject has transfusion-dependent β-thalassemia. In some embodiments, a red blood cell response includes a reduction in red blood cell input of at least 1, 2, 3, 4, or more units in a subject with transfusion-dependent β-thalassemia. In some embodiments, a red blood cell response includes a reduction in red blood cell input of at least 1, 2, 3, 4, or more units in a subject lasting at least 8, 9, 10, 11, 12, 13, 14, 15, 16 weeks, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, a red blood cell response includes a reduction of at least 2 units of red blood cells in a subject for at least 12 weeks, where the subject has transfusion-dependent β-thalassemia. In some embodiments, a red blood cell response includes (i) a reduction in the subject's transfusion burden of at least 33% for at least 12 weeks, and (ii) a reduction of at least 2 units of red blood cells in a subject for at least 12 weeks, where the subject has transfusion-dependent β-thalassemia. In some implementations, the reduction in transfusion burden is compared to the baseline transfusion burden of the subject at 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein.In some embodiments, the reduction in red blood cell units is compared to the number of red blood cells administered to the subject within 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRIIB signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0083] In some embodiments, treatment of a subject according to the methods provided herein (e.g., the subject described in Section 7.5) induces a erythrocyte response in the subject. In some embodiments, the erythrocyte response includes an increase in the subject's hemoglobin concentration greater than 0.75 g / dL, 1 g / dL, 1.25 g / dL, or 1.5 g / dL compared to the subject's hemoglobin concentration prior to treatment according to the methods provided herein, wherein the hemoglobin concentration is measured by the subject's hemoglobin concentration in the subject for at least 12 consecutive weeks without transfusion, and wherein the subject has transfusion-independent β-thalassemia. In some embodiments, the erythrocyte response includes an increase in the subject's hemoglobin concentration greater than 1 g / dL compared to the subject's hemoglobin concentration prior to treatment according to the methods provided herein, wherein the hemoglobin concentration is measured by the subject's hemoglobin concentration in the subject for at least 12 consecutive weeks without transfusion, and wherein the subject has transfusion-independent β-thalassemia. In some embodiments, an ActRII signaling inhibitor is used as described in Section 7.6. In some embodiments, the ActRII signal transduction inhibitor is the ActRII signal transduction inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0084] In some embodiments, transfusion-dependent β-thalassemia patients treated according to the methods provided herein do not require red blood cell transfusions for at least 8, 9, 10, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year after treatment. In some embodiments, transfusion-dependent β-thalassemia patients treated according to the methods provided herein do not require red blood cell transfusions for at least 8 weeks after treatment. In some embodiments, transfusion-dependent β-thalassemia patients treated according to the methods provided herein do not require red blood cell transfusions for at least 12 weeks after treatment. In some embodiments, transfusion-dependent β-thalassemia patients treated according to the methods provided herein do not require red blood cell transfusions for at least 8 weeks after treatment. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0085] In some embodiments, treatment of subjects (e.g., those described in Section 7.5) with the methods provided herein results in a reduction of liver iron concentration of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to the liver iron concentration levels of subjects within 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, the reduction of liver iron concentration in subjects is approximately 10% compared to the liver iron concentration levels of subjects within 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, the liver iron concentration of the subjects is reduced by about 15% compared to the liver iron concentration of the subjects within 1, 2, 3, or 4 weeks prior to starting treatment according to the methods provided herein. In some embodiments, the liver iron concentration of the subjects is reduced by about 20% compared to the liver iron concentration of the subjects within 1, 2, 3, or 4 weeks prior to starting treatment according to the methods provided herein. In some embodiments, the liver iron concentration of the subjects is reduced by between 5% and 30% compared to the liver iron concentration of the subjects within 1, 2, 3, or 4 weeks prior to starting treatment according to the methods provided herein. In some embodiments, the liver iron concentration is reduced by between 10% and 30% compared to the liver iron concentration of the subjects within 1, 2, 3, or 4 weeks prior to starting treatment according to the methods provided herein. In some embodiments, the liver iron concentration is determined according to the assay method described in Section 7.7. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signal transduction inhibitor is the ActRII signal transduction inhibitor 7.6.2 described in Section 7.6.2. In some embodiments, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0086] In some embodiments, treatment of subjects (e.g., subjects described in Section 7.5) according to the methods provided herein results in a reduction of myocardial iron concentration by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to myocardial iron concentrations within 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, myocardial iron concentration is determined according to the assay described in Section 7.7. In some embodiments, ActRII signaling inhibitors are used as described in Section 7.6. In some embodiments, the ActRII signal transduction inhibitor is the ActRII signal transduction inhibitor 7.6.2 described in Section 7.6.2. In some embodiments, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0087] In some embodiments, treating a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in a reduction in the subject's daily iron chelation therapy, such as a decrease in the dose or frequency of administering the subject one or more iron chelation treatments. Non-limiting examples of iron chelation treatments include deferasirox, deferiphenone, and deferipromine. In some embodiments, an ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, an ActRII signaling inhibitor is an ActRII signaling inhibitor as described in Section 7.6.2. In some embodiments, an ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0088] In some embodiments, treatment of subjects (e.g., subjects described in Section 7.5) according to the methods provided herein results in a reduction of serum ferritin levels of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to serum ferritin levels in the subjects at 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, serum ferritin levels are determined according to the assay described in Section 7.7. In some embodiments, ActRII signaling inhibitors are used as described in Section 7.6. In some embodiments, the ActRII signal transduction inhibitor is the ActRII signal transduction inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0089] In some embodiments, treatment of subjects according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in an increase in fetal hemoglobin concentration of the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, or at least 500%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, or at most 500% compared to the subject's fetal hemoglobin concentration 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, the fetal hemoglobin concentration and serum ferritin level are determined according to the assay described in Section 7.7. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO: 25).

[0090] In some embodiments, treatment of subjects according to the methods provided herein (e.g., the subjects described in Section 7.5) results in a reduction of GDF11 concentration by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, or at least 500%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, or at most 500%, compared to the subject's GDF11 concentration at 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, serum ferritin levels of GDF11 are determined according to the assay described in Section 7.7. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0091] In some embodiments, the symptoms associated with one or more clinical complications of β-thalassemia are reduced in subjects treated according to the methods provided herein (e.g., those described in Section 7.5) compared to symptoms experienced 1, 2, 3, or 4 weeks prior to treatment according to the methods provided herein. In some embodiments, the symptoms associated with one or more clinical complications of transfusion-dependent β-thalassemia are reduced in subjects treated according to the methods provided herein (e.g., those described in Section 7.5). Non-limiting examples of transfusion-dependent β-thalassemia include growth arrest, pallor, jaundice, poor muscular system, genu valgum, hepatosplenomegaly, leg ulcers, masses due to extramedullary hematopoiesis, skeletal changes resulting from bone marrow amplification, and clinical complications of prolonged red blood cell transfusions (e.g., hepatitis B virus infection, hepatitis C virus infection) and human immunodeficiency virus infection, allogeneic immunity, and organ damage due to iron overload, such as liver damage, cardiac damage, and endocrine gland damage. In some embodiments, ActRII signaling inhibitors are used as described in Section 7.6. In some embodiments, the ActRII signal transduction inhibitor is the ActRII signal transduction inhibitor 7.6.2 described in Section 7.6.2. In some embodiments, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0092] In some implementations, treatment of a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) reduces symptoms associated with one or more transfusion-dependent β-thalassemia clinical complications compared to symptoms experienced by the subject 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. Non-limiting examples of transfusion-dependent β-thalassemia include endocrine abnormalities such as diabetes mellitus, hypothyroidism, hypogonadism, thrombotic events, pulmonary hypertension, hypercoagulability, transfusion dependence late in life, ineffective erythropoiesis, bone marrow extramedullary hematopoietic tissue expansion, extramedullary hematopoietic mass formation, skeletal deformities, osteopenia, osteoporosis, bone pain, gallstones, leg ulcers, and alloimmunity. In some implementations, an ActRII signaling inhibitor is as described in Section 7.6. In some implementations, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0093] In some implementations, treatment of a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) improves the subject's red blood cell morphology compared to the subject's red blood cell morphology 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. Non-limiting determinants of improved red blood cell morphology include a reduction in the ratio of abnormal red blood cells to the total red blood cell count, a reduction in the ratio of basophilic stippled red blood cells to the total red blood cell count, a reduction in the ratio of atypical red blood cells to the total red blood cell count, a reduction in the ratio of cleaved cells to the total red blood cell count, and a reduction in the ratio of irregularly contracted red blood cells to the total red blood cell count. In some implementations, treating a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in a reduction in the ratio of the subject's abnormal red blood cell count to the subject's total red blood cell count by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to the ratio of the subject's abnormal red blood cell count to the subject's total red blood cell count 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, treatment of subjects according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in a reduction of the subject's basophilic stippling red blood cell count to the subject's total red blood cell count by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to the ratio of the subject's basophilic stippling red blood cell count to the subject's total red blood cell count 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein.In some implementations, treatment of a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in a reduction of the ratio of atypical red blood cells to the total red blood cells by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to the ratio of atypical red blood cells to the total red blood cells of the subject 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some implementations, treatment of a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in a reduction of the ratio of cleavage cell count to total red blood cell count by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to the ratio of cleavage cell count to total red blood cell count in the subject 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, treatment of a subject according to the methods provided herein (e.g., the subject described in Section 7.5) results in a reduction of the ratio of irregularly contracting red blood cells to the total red blood cell count by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at most 100%, compared to the ratio of irregularly contracting red blood cells to the total red blood cell count in the subject during 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, the serum ferritin level is determined according to the assay described in Section 7.7. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2.In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0094] In some embodiments, treatment of subjects according to the methods provided herein (e.g., the subjects described in Section 7.5) results in a reduction of one, two, three, four, or more symptoms of osteoporosis within one, two, three, or four weeks prior to initiating treatment according to the methods provided herein. In some embodiments, treatment of subjects according to the methods provided herein (e.g., the subjects described in Section 7.5) results in a reduction of one, two, three, four, or more symptoms of osteopenia within one, two, three, or four weeks prior to initiating treatment according to the methods provided herein. In some embodiments, treatment of a subject according to the methods provided herein (e.g., subject 7.5 as described in Section 7.5) results in an increase in bone mineral density of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, or at least 500%, or at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, or at most 500%, compared to the subject's bone mineral density 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, bone mineral density is total body bone mineral density, total hip bone mineral density, or lumbar spine bone mineral density. In some embodiments, bone mineral density is determined according to the assay method described in Section 7.7. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO: 25).

[0095] In some embodiments, treatment of a subject according to the methods provided herein (e.g., the subject described in Section 7.5) results in a reduction of skeletal deformities compared to those occurring 1, 2, 3, or 4 weeks prior to initiating treatment according to the methods provided herein. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO: 25).

[0096] In some embodiments, treatment of a subject using the methods provided herein (e.g., the subject described in Section 7.5) results in an improvement in the subject's quality of life compared to the quality of life of a subject within 1, 2, 3, or 4 weeks prior to initiating treatment using the methods provided herein. In some embodiments, quality of life is measured according to the assay described in Section 7.7. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0097] 7.3.2 Adjusting the dosage for administration This document also provides methods for treating β-thalassemia in subjects in need (see Section 7.3.1, 7.3.1), methods comprising analyzing the subject's hemoglobin concentration as a means of determining the subsequent dosing regimen for the subject. In some embodiments, the subject's hemoglobin concentration may be used to (i) evaluate the appropriate dosing for the subject, wherein the subject is a candidate for or currently being treated with an ActRII signaling inhibitor (e.g., activin ligand trap); (ii) evaluate whether the dose of the ActRII signaling inhibitor should be adjusted during treatment; and / or (iii) evaluate the appropriate maintenance dose of the ActRII signaling inhibitor. Dosing with the ActRII signaling inhibitor may be initiated, increased, decreased, delayed, or terminated based on the subject's hemoglobin concentration. See, for example, Tables 1 and 2. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0098] Table 1. Subsequent dosing regimens: dose extension, dose reduction, and dose cessation Table 2. Starting Dose Levels and Dose Decreases and Increases In some embodiments, the method of treating a subject with β-thalassemia (see Section 7.3.1, 7.3.1) further includes (a) performing a first measurement of the subject's hemoglobin concentration; (b) performing a second measurement of the subject's hemoglobin concentration after a first period of time; and (c) after the second period of time, terminating the initial dose and administering a subsequent dose of an ActRII signaling inhibitor to the subject, wherein the subsequent dose is administered subcutaneously in the subject's upper arm, abdomen, or thigh. In some embodiments, the method further includes performing a third measurement of the subject's hemoglobin concentration. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6, 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0099] In some embodiments, the first and / or second measurements are performed as described in Section 7.7. In some embodiments, the first measurement of hemoglobin concentration is performed before administering the initial dose of the ActRII signaling inhibitor to the subject. In some embodiments, the first measurement of hemoglobin concentration is immediately following or within up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week after administering the initial dose of the ActRII signaling inhibitor to the subject. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor 7.6.2 described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0100] In some implementations, a second measurement of hemoglobin concentration is performed approximately 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the initial dose of the ActRII signaling inhibitor is administered to the subject.

[0101] In some implementations, the second period is within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks when the second measurement is performed.

[0102] In some embodiments, the subsequent dose of the ActRII signaling inhibitor is about 0.3 mg / kg, about 0.45 mg / kg, about 0.6 mg / kg, about 1.0 mg / kg, or about 1.25 mg / kg. In some embodiments, the subsequent dose of the ActRII signaling inhibitor is titrated up to a maximum subsequent dose of about 1.25 mg / kg. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0103] In some embodiments, the method further includes performing a third measurement of the subject's hemoglobin concentration.

[0104] In one specific embodiment, (a) the second measurement of hemoglobin concentration is less than or equal to 12.5 g / dL; (b) the second measurement of hemoglobin concentration is greater than the first measurement of hemoglobin concentration which is less than or equal to 1.5 g / dL; and (c) the subsequent dose is equal to the initial dose. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0105] In another specific embodiment, (a) the second measurement of hemoglobin concentration is less than or equal to 12.5 g / dL; (b) the second measurement of hemoglobin concentration is greater than the first measurement of hemoglobin concentration by more than 1.5 g / dL; and (c) the subsequent dose is less than about 25% of the initial dose. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0106] In yet another specific embodiment, (a) a second measurement of hemoglobin concentration (i) is greater than 12.5 g / dL and less than or equal to 14 g / dL; and (ii) is greater than the first measurement of hemoglobin concentration but less than or equal to 1.5 g / dL; (b) subsequent doses are equal to the initial dose; and (c) a second period includes a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is less than or equal to 12.5 g / dL. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0107] In yet another specific embodiment, (a) a second measurement of hemoglobin concentration (i) is greater than 12.5 g / dL and less than or equal to 14 g / dL, and (ii) is greater than 1.5 g / dL greater than the first measurement of hemoglobin concentration; (b) a subsequent dose is less than about 25% of the initial dose; and (c) a second period comprising a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is determined to be (i) less than or equal to 12.5 g / dL, and (ii) the change in hemoglobin concentration between the first and third measurements is less than or equal to 1.5 g / dL. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor 7.6.2 described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0108] In yet another specific embodiment, (a) a second measurement of hemoglobin concentration is greater than 14 g / dL; (b) a subsequent dose is less than approximately 25% of the initial dose; and (c) a second period includes a dose extension of up to 12 weeks until a third measurement of hemoglobin concentration is less than 12.5 g / dL. In some embodiments, the method further includes determining a third measurement of hemoglobin concentration. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0109] In some embodiments, the initial dose is administered as described in Section 7.4. In some embodiments, the initial dose is administered to the subject every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the initial dose is administered to the subject via subcutaneous injection. In some embodiments, the initial dose is administered to the subject in the upper arm, abdomen, or thigh. In some embodiments, the initial dose is administered to the subject via subcutaneous injection in the upper arm, abdomen, or thigh every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0110] In some embodiments, the initial dose is administered as described in Section 7.4. In some embodiments, the initial dose is administered to the subject every 21 days. In some embodiments, the initial dose is administered to the subject via subcutaneous injection. In some embodiments, the initial dose is administered to the subject in the upper arm, abdomen, or thigh. In some embodiments, the initial dose is administered to the subject via subcutaneous injection in the upper arm, abdomen, or thigh every 21 days. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor 7.6.2 described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0111] In some embodiments, the initial dose is administered as described in Section 7.4. In some embodiments, the initial dose is administered to the subject every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the initial dose is administered to the subject via subcutaneous injection. In some embodiments, the initial dose is administered to the subject in the upper arm, abdomen, or thigh. In some embodiments, the initial dose is administered to the subject via subcutaneous injection in the upper arm, abdomen, or thigh every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0112] In some embodiments, the subsequent dose is administered as described in Section 7.4. In some embodiments, the subsequent dose is administered to the subject every 21 days. In some embodiments, the subsequent dose is administered to the subject via subcutaneous injection. In some embodiments, the subsequent dose is administered to the subject in the upper arm, abdomen, or thigh. In some embodiments, the subsequent dose is administered to the subject via subcutaneous injection in the upper arm, abdomen, or thigh every 21 days. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRIIB signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0113] In some embodiments, subsequent doses are administered as described in Section 7.4. In some embodiments, subsequent doses are administered to the subject every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, subsequent doses are administered to the subject via subcutaneous injection. In some embodiments, subsequent doses are administered to the subject in the upper arm, abdomen, or thigh. In some embodiments, subsequent doses are administered to the subject via subcutaneous injection in the upper arm, abdomen, or thigh every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some implementations, the ActRIIB signal transduction inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25).

[0114] In some other embodiments, the subject is the subject described in Section 7.5. In some embodiments, the subject has β-thalassemia. In some embodiments, the subject has transfusion-dependent β-thalassemia. In some embodiments, the subject has severe β-thalassemia. In some embodiments, transfusion-dependent β-thalassemia is severe β-thalassemia. In some embodiments, the subject has non-transfusion-dependent β-thalassemia. In some embodiments, the subject has moderate β-thalassemia. In some embodiments, non-transfusion-dependent β-thalassemia is moderate β-thalassemia.

[0115] In some embodiments, hemoglobin concentrations (i.e., first hemoglobin concentration, second hemoglobin concentration, and third hemoglobin concentration) are determined as described in Section 7.7.

[0116] In some implementations, the methods provided herein are used in combination with a second pharmaceutical active agent or therapy as described in Section 7.8.

[0117] In some embodiments, the ActRII signaling inhibitor is as described in Section 7.6. In some embodiments, the ActRII signaling inhibitor is the ActRII signaling inhibitor described in Section 7.6.2. In some embodiments, the ActRII signaling inhibitor is ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25). In some embodiments, the ActRII signaling inhibitor is the ActRIIA signaling inhibitor described in Section 7.6.1. In some embodiments, the ActRII signaling inhibitor is ActRIIA-Fc, such as ActRIIA-hFc (e.g., SEQ ID NO:7).

[0118] 7.4 Dosing regimen In some embodiments, the ActRII signaling inhibitor administered according to the methods provided herein (see Sections 7.3.1 and 7.3.2) is at a dose of about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, or about 1.2 mg / kg. In some embodiments, the ActRII signaling inhibitor administered according to the methods provided herein (see Sections 7.3.1 and 7.3.2) is at a dose of about 0.8 mg / kg. In some embodiments, the ActRII inhibitor is an inhibitor of ActRIIB signaling listed in Section 7.6.2. In some embodiments, the ActRII signaling inhibitor is an ActRIIB-Fc, such as ActRIIB-hFc (e.g., SEQ ID NO:25). In some embodiments, the ActRII signaling inhibitor is an inhibitor of ActRIIA signaling listed in Section 7.6.1. In some embodiments, the ActRII signal transduction inhibitor is an ActRIIA-Fc, such as ActRIIA-hFc (e.g., SEQ ID NO: 7). In some embodiments, the ActRII signal transduction inhibitor is a combination of an ActRIIA signal transduction inhibitor and an ActRIIB signal transduction inhibitor.

[0119] In some embodiments, the ActRII signaling inhibitor is administered subcutaneously to the subject. In some embodiments, the ActRII signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh. In some embodiments, the ActRII signaling inhibitor is administered to the subject every 21 days. In some embodiments, the ActRII signaling inhibitor is administered to the subject every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the ActRII signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days. In some embodiments, the ActRII signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0120] In some embodiments, the ActRII signal transduction inhibitor is the composition 7.9 described in Section 7.9. In some embodiments, the ActRII signal transduction inhibitor is a sterile, preservative-free lyophilized powder reconstituted in water for injection. In some embodiments, a single dose of the ActRII signal transduction inhibitor is reconstituted in a volume greater than 1 mL of water for injection. In these embodiments, a single dose of the ActRII signal transduction inhibitor is administered to the subject via two injections of equal volumes of the reconstituted ActRII signal transduction inhibitor. In some embodiments, the two injections are administered to the subject at different sites, for example, one injection in the right leg and one injection in the left leg.

[0121] In some embodiments, the dose of the ActRII signaling inhibitor is an initial dose. In some embodiments, the initial dose is approximately 0.8 mg / kg.

[0122] In some embodiments, the dose of the ActRII signaling inhibitor is a subsequent dose. In some embodiments, the subsequent dose is greater than the initial dose. In some embodiments, the subsequent dose is less than the initial dose. In some embodiments, the subsequent dose is about 0.3 mg / kg, about 0.45 mg / kg, about 0.6 mg / kg, about 1.0 mg / kg, or about 1.25 mg / kg. In some embodiments, the subsequent dose is about 0.3 mg / kg, about 0.45 mg / kg, about 0.6 mg / kg, about 1.0 mg / kg, or about 1.25 mg / kg. In some embodiments, the subsequent dose is about 0.3 mg / kg. In some embodiments, the subsequent dose is about 0.45 mg / kg. In some embodiments, the subsequent dose is about 0.6 mg / kg. In some embodiments, the subsequent dose is about 1.0 mg / kg. In some embodiments, the subsequent dose is about 1.25 mg / kg. In some embodiments, the subsequent dose is greater than the initial dose of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or greater than the initial dose of about 35 mg, or about 0.05 mg / kg, about 0.1 mg / kg, about 0.15 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, or about 0.5 mg / kg.

[0123] In some implementations, subsequent doses of the ActRII signaling inhibitor are administered at intervals and amounts sufficient to achieve serum concentrations of about 0.2 μg / kg or greater, and serum levels of about 1 μg / kg or 2 μg / kg or greater are required to achieve a significant effect on bone mineral density and strength. Subsequent dosing regimens can be designed to achieve serum concentrations between 0.2 and 15 μg / kg and optionally between 1 and 5 μg / kg. In humans, a serum level of 0.2 μg / kg can be achieved with a single subsequent dose of about 0.1 mg / kg or greater, and a serum level of 1 μg / kg can be achieved with a single subsequent dose of about 0.3 mg / kg or greater. The observed serum half-life of the molecule is between about 20 and 30 days, significantly longer than that of most Fc fusion proteins, thus allowing for sustained effective serum levels to be achieved, for example, on a weekly or bi-weekly basis, by administration of about 0.2–0.4 mg / kg, or by using higher doses with longer intervals between administrations. For example, subsequent doses of approximately 1-3 mg / kg can be used on a monthly or bimonthly basis, requiring only dosing every 3, 4, 5, 6, 9, 12, or more months, with sufficiently sustained effects on bone. Serum levels of ActRII signaling inhibitors can be measured by any method known to a person skilled in the art. For example, serum levels of ActRII signaling inhibitors can be determined using an antibody against the ActRII signaling inhibitor, such as an ELISA.

[0124] In some embodiments, subsequent doses are administered more frequently than the initial dose. In some embodiments, subsequent doses are administered less frequently than the initial dose. In some embodiments, subsequent doses are administered at the same frequency as the initial dose. In some embodiments, subsequent doses are administered every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, subsequent doses are administered every 21 days. In some embodiments, subsequent doses are administered continuously and / or indefinitely.

[0125] When used in combination with the dosages provided herein (e.g., the dosage of an ActRII signaling inhibitor or a second active agent), the term “about” means any value within 1, 5, or 10% of the cited value.

[0126] 7.5 Patient Group Subjects treated according to the methods described herein can be any mammal, such as rodents and primates, and in a preferred embodiment, humans. In some embodiments, the methods described herein can be used to treat subjects with β-thalassemia, such as transfusion-dependent β-thalassemia, non-transfusion-dependent β-thalassemia, severe β-thalassemia, and moderate β-thalassemia, to reduce the transfusion burden on subjects with β-thalassemia, or to monitor the treatment, and / or, in any mammal, such as rodents or primates, and in a preferred embodiment, in human subjects, to select subjects to be treated according to the methods provided herein.

[0127] In some implementations, the subjects treated according to the methods described herein can be of any age. In some implementations, the subjects treated according to the methods described herein are under 18 years of age. In one specific implementation, the subjects treated according to the methods described herein are under 13 years of age. In another specific implementation, the subjects treated according to the methods described herein are under 12 years of age, under 11 years of age, under 10 years of age, under 9 years of age, under 8 years of age, under 7 years of age, under 6 years of age, or under 5 years of age. In another specific implementation, the subjects treated according to the methods described herein are 1-3 years of age, 3-5 years of age, 5-7 years of age, 7-9 years of age, 9-11 years of age, 11-13 years of age, 13-15 years of age, 15-20 years of age, 20-25 years of age, 25-30 years of age, or over 30 years of age. In another specific implementation, the subjects treated according to the methods described herein are 30-35 years of age, 35-40 years of age, 40-45 years of age, 45-50 years of age, 50-55 years of age, 55-60 years of age, or over 60 years of age. In another specific implementation, the subjects treated according to the method described herein are 60-65 years old, 65-70 years old, 70-75 years old, 75-80 years old, or older than 80 years old.

[0128] In some embodiments, the subject treated according to the methods described herein (see Section 7.3) has β-thalassemia. In some embodiments, the β-thalassemia is transfusion-dependent β-thalassemia. Transfusion-dependent β-thalassemia is also known as “Cooley's anemia”. In some embodiments, the β-thalassemia is severe β-thalassemia. In some embodiments, transfusion-dependent β-thalassemia is severe β-thalassemia. In some embodiments, the β-thalassemia is non-transfusion-dependent β-thalassemia. In some embodiments, the β-thalassemia is moderate β-thalassemia. In some embodiments, transfusion-dependent β-thalassemia is moderate non-β-thalassemia. In some embodiments, the subject has HbE / β-thalassemia. In some embodiments, the subject (i) has severe β-thalassemia; (ii) has severe HbE / β-thalassemia; and (iii) is transfusion-dependent. In some implementations, the subject (i) has moderate β-thalassemia; (ii) has mild / moderate HbE / β-thalassemia; and (iii) is transfusion-free.

[0129] In some embodiments, the subject treated according to the methods described herein (see Section 7.3) has transfusion-dependent β-thalassemia. In some embodiments, the subject is diagnosed with transfusion-dependent β-thalassemia. In some embodiments, the subject is diagnosed with β-thalassemia and hemoglobin E. In some embodiments, the diagnosis is confirmed by genetic analysis. In some embodiments, the transfusion-dependent β-thalassemia is severe β-thalassemia. In some embodiments, the transfusion-dependent β-thalassemia is severe β-thalassemia. In some embodiments, the subject possesses a phenotype of homozygosity or complex heterozygosity containing a mutant β-globin allele. In some embodiments, homozygosity includes β... 0 / β 0 , where β 0 This refers to alleles associated with a deficiency in β-globin chain synthesis. In some implementations, homozygosity includes β... + / β + , where β + This refers to alleles associated with reduced β-globin chain synthesis. In some implementations, complex heterozygosity includes β... 0 / β + , where β 0 This refers to alleles associated with a deficiency in β-globin chain synthesis, and among them, β... + This refers to alleles associated with reduced β-globin chain synthesis. In some implementations, complex heterozygosity includes β... 0 / HbE, where β 0This refers to the allele associated with a deficiency in β-globin chain synthesis, where HbE refers to hemoglobin E. In some embodiments, complex heterozygosity includes β-globin chain synthesis. + / HbE, where β + This refers to an allele associated with reduced β-globin chain synthesis, where HbE refers to hemoglobin E. In some embodiments, the subject has symptomatic thalassemia. In some embodiments, the subject has a co-inherited duplication of the α-globin gene. In some embodiments, the subject has been diagnosed with transfusion-dependent β-thalassemia. In some embodiments, the diagnosis is confirmed by genetic analysis. In some embodiments, the subject is a human infant. In some embodiments, the subject has hereditary fetal hemoglobin persistence.

[0130] In some implementations, the subject requires regular, lifelong red blood cell transfusions. In some implementations, a subject with transfusion-dependent β-thalassemia requires more than 5 units of red blood cells over a 24-week period. In some implementations, a subject with transfusion-dependent β-thalassemia requires more than 6 units of red blood cells over a 24-week period. In some implementations, the subject has a high transfusion burden. In some implementations, a high transfusion burden is 12 or more units of red blood cells within the 24 weeks prior to treatment according to the methods provided herein. In some implementations, the subject has a low transfusion burden. In some implementations, a low transfusion burden is 7-12 units of red blood cells within the 24 weeks prior to treatment according to the methods provided herein.

[0131] In some implementations, the subject suffers from one or more clinical complications of transfusion-dependent β-thalassemia. Non-limiting examples of clinical complications of transfusion-dependent β-thalassemia include growth arrest, pallor, jaundice, poor muscular development, genu valgum, hepatosplenomegaly, leg ulcers, masses due to extramedullary hematopoiesis, and skeletal changes resulting from bone marrow amplification. In some implementations, the subject suffers from one or more complications of long-term red blood cell transfusion. Non-limiting examples of complications of long-term red blood cell transfusion include transfusion-related infections such as hepatitis B virus infection, hepatitis C virus infection, and human immunodeficiency virus infection, allogeneic immunity, and organ damage due to iron overload, such as liver damage, cardiac damage, and endocrine gland damage.

[0132] In some embodiments, the subjects treated according to the methods described herein (see Section 7.3) have transfusion-independent β-thalassemia. In some embodiments, subjects with transfusion-independent β-thalassemia require 0-5 units of red blood cells over 24 weeks. In some embodiments, subjects with transfusion-independent β-thalassemia require 0-6 units of red blood cells over 24 weeks. In some embodiments, the subject is diagnosed with β-thalassemia. In some embodiments, the subject is diagnosed with both β-thalassemia and hemoglobin E. In some embodiments, β-thalassemia is confirmed by genetic analysis. In some embodiments, the transfusion-independent β-thalassemia is moderate β-thalassemia. In some embodiments, the transfusion-independent β-thalassemia is mild-to-moderate hemoglobin E / β-thalassemia. In some embodiments, transfusion-independent β-thalassemia does not require regular red blood cell transfusions. In some embodiments, the subject rarely requires red blood cell transfusions. In some implementations, transfusion-independent β-thalassemia requires regular red blood cell transfusions later in life. In some implementations, the subject received 0-5 units of red blood cells within 24 weeks prior to treatment according to the methods described herein. In some implementations, the subject received 0-6 units of red blood cells within 24 weeks prior to treatment according to the methods described herein. In some implementations, the subject has a mean baseline hemoglobin level of less than 10.0 g / dL.

[0133] In some embodiments, β-thalassemia is transfusion-free β-thalassemia. In some embodiments, β-thalassemia is moderate β-thalassemia. In some embodiments, transfusion-dependent β-thalassemia is moderate non-β-thalassemia. In some embodiments, the subject includes a phenotype containing complex heterozygosity. In some embodiments, complex heterozygosity includes β... 0 Alleles, of which β 0 This refers to alleles associated with a deficiency in β-globin chain synthesis. In some implementations, complex heterozygosity includes β... + Alleles, of which β + This refers to alleles associated with reduced β-globin chain synthesis. In some implementations, complex heterozygosity includes β... 0 / β + , where β 0 This refers to alleles associated with a deficiency in β-globin chain synthesis, and among them, β... +This refers to alleles associated with reduced β-globin chain synthesis. In some embodiments, complex heterozygosity includes one or more hemoglobin variants. In some embodiments, the hemoglobin variant is hemoglobin E. In some embodiments, the subject (i) has a co-inherited phenotype containing two severe β-globin chain mutations, and (ii) suffers from α-thalassemia. In some embodiments, the subject (i) has a co-inherited phenotype containing two severe β-globin chain mutations, and (ii) suffers from hereditary fetal hemoglobin persistence. In some embodiments, the subject has symptomatic thalassemia. In some embodiments, the subject has a co-inherited duplication of the α-globin gene. In some embodiments, the subject is diagnosed with β-thalassemia. In some embodiments, the diagnosis is confirmed by genetic analysis.

[0134] In some implementations, the subject exhibits one or more transfusion-independent clinical complications of β-thalassemia. Non-limiting examples of transfusion-independent clinical complications of β-thalassemia include endocrine abnormalities such as diabetes mellitus, hypothyroidism, hypogonadism, thrombotic events, pulmonary hypertension, hypercoagulability, transfusion dependence late in life, ineffective erythropoiesis, extramedullary hematopoietic tissue expansion in the bone marrow, extramedullary hematopoietic mass formation, skeletal deformities, osteopenia, osteoporosis, bone pain, gallstones, and leg ulcers. In some implementations, the subject exhibits allogeneic immunity.

[0135] In some implementations, the subject exhibits mild symptoms of β-thalassemia. In some implementations, the subject has almost normal growth.

[0136] In some implementations, non-transfusion-dependent β-thalassemia subjects exhibit severe symptoms. Non-limiting examples of severe symptoms include growth arrest, developmental delay, and skeletal deformities.

[0137] In some implementations, the subject has splenomegaly. In some implementations, the splenomegaly occurs during the first 6-12 months of the subject's life.

[0138] In some implementations, the object experiences growth impairment during the first 10 years of its life.

[0139] In some embodiments, the subject exhibits microcytic, low-chromosome-index anemia. In some embodiments, the subject's hemoglobin A2 level is elevated before treatment using the methods provided herein, compared to hemoglobin A2 levels in a reference population (e.g., the reference population described in Section 7.7). In some embodiments, the subject's fetal hemoglobin level is elevated before treatment using the methods provided herein, compared to fetal hemoglobin levels in a reference population (e.g., the reference population described in Section 7.7).

[0140] In some implementations, the object does not express hemoglobin S.

[0141] In some implementations, the subject does not express hemoglobin S. In some implementations, the subject has not received red blood cell transfusions within 12 weeks prior to treatment according to the methods described herein, and the subject has non-transfusion-dependent β-thalassemia. In some implementations, the subject does not have active hepatitis C infection. In some implementations, the subject does not have active hepatitis B infection. In some implementations, the subject is not positive for human immunodeficiency virus. In some implementations, the subject does not have insulin-dependent diabetes mellitus. In some implementations, the subject has not received erythropoiesis stimulants within 3 months prior to treatment according to the methods described herein. In some implementations, the subject has not received iron chelation therapy within 168 days prior to treatment according to the methods described herein. In some implementations, the subject has not received hydroxyurea therapy within 168 days prior to treatment according to the methods described herein. In some implementations, the subject has not received bisphosphonates within 168 days prior to treatment according to the methods described herein. In some implementations, the subject does not have uncontrolled hypertension. According to NCI CTCAE version 4.0, uncontrolled hypertension is defined as grade >1. In some implementations, the subject does not have liver disease with ALT greater than 3 times the upper limit of normal. In some implementations, the subject does not have liver disease with histopathological evidence of cirrhosis / fibrosis determined by liver biopsy. In some implementations, the subject does not have heart disease. Heart disease or heart failure may be classified as Grade 3 or higher according to the New York Heart Association. In some implementations, the subject does not have arrhythmias requiring treatment. In some implementations, the subject does not have lung disease. Non-limiting examples of lung disease include pulmonary fibrosis and pulmonary hypertension. In some implementations, the subject's creatinine clearance is not less than 60 mL / min as determined by the Cockroff-Gault method. In some implementations, the subject does not have folate deficiency. In some implementations, the subject does not have Grade 3 or higher proteinuria. In some implementations, the subject does not have adrenal insufficiency. In some implementations, the subject has not undergone major surgery within 30 days prior to treatment with the methods provided herein, with the exception of splenectomy. In some implementations, the subject does not have a history of severe allergic or anaphylactic reactions or hypersensitivity to recombinant proteins. In some implementations, the subject has not received long-term anticoagulation therapy. Non-limiting examples of anticoagulant therapy include heparin and warfarin. In some embodiments, the subject has not received treatment with cytotoxic agents, systemic corticosteroids, immunosuppressants, or anticoagulant therapy within 28 days prior to treatment according to the methods provided herein.

[0142] In some implementations, the subject is undergoing other therapeutic interventions. Non-limiting examples of other therapeutic interventions include splenectomy, blood transfusion therapy, iron chelation therapy, and fetal hemoglobin inducers. In some implementations, the subject requires iron chelation therapy. See Section 7.8 for a description of combination therapies.

[0143] In some implementations, the object is the object described in Section 8.

[0144] As used in this article, the terms "patient" and "object" are used interchangeably.

[0145] 7.6 Inhibitors of ACTRII signaling The ActRII signal transduction inhibitors described in this section and known in the art can be used in the methods provided herein. In some embodiments, the ActRII signal transduction inhibitors described in this section can be used in the methods provided herein (see Section 7.3).

[0146] The inhibitory receptors of ACTRII signaling included herein are ActRIIA signaling inhibitors and ActRIIB signaling inhibitors (see below). In some embodiments, the ActRII signaling inhibitor is specific for ActRIIA signaling. In other embodiments, the ActRII signaling inhibitor is specific for ActRIIB signaling. In some embodiments, the ActRII signaling inhibitor preferentially inhibits ActRIIA signaling. In other embodiments, the ActRII signaling inhibitor preferentially inhibits ActRIIB signaling. In some embodiments, the ActRII signaling inhibitor inhibits both ActRIIA and ActRIIB signaling.

[0147] In some embodiments, the inhibitor of ACTRII signaling can be a polypeptide containing the activin-binding domain of ActRII. While not bound by theory, such activin-binding domains contain the polypeptide's multivalent chelating agent activin, thus blocking activin signaling. The polypeptide containing these activin-binding domains may contain all or part of the extracellular domain of ActRII (i.e., all or part of the extracellular domain of ActRIIA or all or part of the extracellular domain of ActRIIB). In a specific embodiment, the extracellular domain of ActRII is soluble.

[0148] In some embodiments, the activin-binding domain of the peptide is linked to the Fc moiety of the antibody (i.e., generating a conjugate containing the activin-binding domain of the peptide containing the ActRII receptor and the Fc moiety of the antibody). While not bound by theory, the antibody moiety confers high stability to the conjugate. In some embodiments, the activin-binding domain is linked to the Fc moiety of the antibody via a linker (e.g., a peptide linker).

[0149] The ACTRII signaling inhibitors used in the compositions and methods described herein comprise molecules that directly or indirectly, extracellularly or intracellularly, inhibit ActRIIA and / or ActRIIB signaling. In some embodiments, the ActRIIA and / or ActRIIB signaling inhibitors used in the compositions and methods described herein inhibit ActRIIA and / or ActRIIB signaling through interaction with the receptor itself. In other embodiments, the ActRIIA and / or ActRIIB signaling inhibitors used in the compositions and methods described herein inhibit ActRIIA and / or ActRIIB signaling through interaction with ActRIIA and / or ActRIIB ligands (e.g., activins).

[0150] 7.6.1 Inhibitors of ACTRIIA signal transduction As used herein, the term "ActRIIA" refers to a family of activin type IIA receptor (ActRIIA) proteins from any species, as well as variants derived from these ActRIIA proteins through mutagenesis or other modifications. References to ActRIIA herein should be understood as referring to any currently identified form. Members of the ActRIIA family are generally transmembrane proteins, consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[0151] ActRIIA signal transduction inhibitors used in the compositions and methods described herein include, but are not limited to, soluble ActRIIA peptides that bind activin; activin-binding peptides (particularly activin A or B subunits, also known as β-activin). A or β BAntibodies that bind to ActRIIA and disrupt ActRIIA binding; antibodies that bind to ActRIIA and disrupt activin binding; non-antibody proteins that bind to activin or ActRIIA (see, for example, WO / 2002 / 088171, WO / 2006 / 055689, WO / 2002 / 032925, WO / 2005 / 037989, US 2003 / 0133939 and US 2005 / 0238646, each of which is incorporated herein by reference in its entirety as examples of such proteins and the design and selection of such proteins); random peptides that bind to activin or ActRIIA and may be conjugated to an Fc domain.

[0152] In some embodiments, two or more different proteins (or other portions) having activin or ActRIIA binding activity, particularly activin conjugates that block type I (e.g., soluble type I activin receptor) and type II (e.g., soluble type II activin receptor) binding sites respectively, can be linked together to produce a bifunctional or multifunctional binding molecule that inhibits ActRIIA signal transduction, and thus can be used in the compositions and methods described herein. In some embodiments, the activin-ActRIIA signal transduction axis antagonist that inhibits ActRIIA signal transduction includes nucleic acid aptamers, small molecules, and other agents used in the compositions and methods described herein.

[0153] 7.6.1.1 ActRIIA signal transduction inhibitors containing ActRIIA peptides The term "ActRIIA polypeptide" includes polypeptides comprising any naturally occurring polypeptide of a member of the ActRIIA family, as well as any variants (including mutants, fragments, fusions, and peptide mimics) that retain their useful activity. For example, ActRIIA polypeptides include polypeptides derived from sequences of any known ActRIIA that have at least about 80% sequence identity with an ActRIIA polypeptide, optionally at least 85%, 90%, 95%, 97%, 98%, 99%, or higher. For example, ActRIIA polypeptides can bind to and inhibit the function of ActRIIA proteins and / or activins. ActRIIA polypeptides can be selected for their ability to promote bone growth and bone mineralization. Examples of ActRIIA polypeptides include human ActRIIA precursor polypeptides (SEQ ID NO:1) and soluble human ActRIIA polypeptides (e.g., SEQ ID NO:2, 3, 7, and 12). For the ActRIIA precursor polypeptide whose amino acid sequence is described in SEQ ID NO:1, the signal peptide of the human ActRIIA precursor polypeptide is located at amino acid positions 1 to 20; the extracellular domain is located at amino acid positions 21 to 135; and the N-glycosylation sites of the human ActRIIA precursor polypeptide (SEQ ID NO:1) are located at amino acid positions 43 and 56 of SEQ ID NO:1. The nucleic acid sequence encoding the human ActRIIA precursor polypeptide of SEQ ID NO:1 is disclosed as SEQ ID NO:4 (nucleotides 164-1705 of Genbank entry NM_001616). The nucleic acid sequence encoding the soluble human ActRIIA polypeptide of SEQ ID NO:2 is disclosed as SEQ ID NO:5. See Table 3 for descriptions of the sequences.

[0154] In specific embodiments, the ActRIIA peptides used in the compositions and methods described herein are soluble ActRIIA peptides. The extracellular domains of ActRIIA proteins can bind activins and are generally soluble, thus they can be referred to as soluble activin-binding ActRIIA peptides. Therefore, as used herein, the term "soluble ActRIIA peptide" generally refers to a peptide containing the extracellular domain of an ActRIIA protein, including any naturally occurring extracellular domain of the ActRIIA protein and any variants thereof (including mutants, fragments, and peptide mimicry forms). Soluble ActRIIA peptides can bind activins; however, wild-type ActRIIA proteins do not exhibit significant selectivity in binding activins to GDF8 / 11. Additional specificity for activins can be conjugated to natural or modified ActRIIA proteins by coupling them with a second activin-selective binding agent. Examples of soluble activin-binding ActRIIA peptides include the soluble peptides exemplified in SEQ ID NO: 2, 3, 7, 12, and 13. Other examples of soluble activin-binding ActRIIA peptides, besides the extracellular domain of the ActRIIA protein, include signal sequences such as bee venom leader sequences (SEQ ID NO:8), tissue plasminogen activator (TPA) leader regions (SEQ ID NO:9), or the native ActRIIA leader region (SEQ ID NO:10). The ActRIIA-hFc peptide exemplified in SEQ ID NO:13 uses the TPA leader region.

[0155] In some embodiments, the inhibitors of ActRIIA signaling used in the compositions and methods described herein comprise conjugates / fusion proteins containing an activin-binding domain of ActRIIA linked to the Fc moiety of an antibody. In some embodiments, the activin-binding domain is linked to the Fc moiety of the antibody via a linker (e.g., a peptide linker). Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In some cases, the Fc domain having one or more of these mutations (e.g., the Asp-265 mutation) has a reduced ability to bind to the Fcγ receptor relative to the wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., the Asn-434 mutation) has an increased ability to bind to the MHC class I-associated Fc receptor (FcRN) relative to the wild-type Fc domain. Exemplary fusion proteins comprising a soluble extracellular domain of ActRIIA fused to an Fc domain are listed in SEQ ID NOs: 6, 7, 12, and 13.

[0156] In one specific embodiment, the ActRIIA signaling inhibitor used in the compositions and methods described herein comprises an extracellular domain or a portion thereof of ActRIIA linked to the Fc portion of an antibody, wherein the ActRIIA signaling inhibitor comprises an amino acid sequence having at least 75% identity with an amino acid sequence selected from SEQ ID NO: 6, 7, 12, and 13. In another specific embodiment, the ActRIIA signaling inhibitor used in the compositions and methods described herein comprises an extracellular domain or a portion thereof of ActRIIA linked to the Fc portion of an antibody, wherein the ActRIIA signaling inhibitor comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 6, 7, 12, and 13.

[0157] In some embodiments, the inhibitors of ActRIIA signaling used in the compositions and methods described herein comprise a truncated form of the extracellular domain of ActRIIA. The truncation may be at the carboxyl terminus and / or amino terminus of the ActRIIA polypeptide. In some embodiments, the length of the truncation relative to the extracellular domain of the mature ActRIIA polypeptide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 N-terminal amino acids of the extracellular domain of the mature ActRIIA polypeptide. In some embodiments, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the extracellular domain of a mature ActRIIA polypeptide. For example, truncated forms of ActRIIA include polypeptides having amino acids 20-119; 20-128; 20-129; 20-130; 20-131; 20-132; 20-133; 20-134; 20-131; 21-131; 22-131; 23-131; 24-131, and 25-131, wherein the amino acid positions refer to the amino acid positions in SEQ ID NO:1.

[0158] In some embodiments, the inhibitors of ActRIIA signaling used in the compositions and methods described herein comprise the extracellular domain of ActRIIA having one or more amino acid substitutions. In some embodiments, the inhibitors of ActRIIA signaling used in the compositions and methods described herein comprise a truncated form of the extracellular domain of ActRIIA also having amino acid substitutions.

[0159] In one specific embodiment, the ActRIIA signaling inhibitor used in the compositions and methods described herein is a fusion protein between the extracellular domain of the human ActRIIA receptor and the Fc moiety of IgG1. In another specific embodiment, the ActRIIA signaling inhibitor used in the compositions and methods described herein is a fusion protein between a truncated extracellular domain of the human ActRIIA receptor and the Fc moiety of IgG1. In yet another specific embodiment, the ActRIIA signaling inhibitor used in the compositions and methods described herein is a fusion protein between a truncated extracellular domain of the human ActRIIA receptor and the Fc moiety of IgG1, wherein the truncated extracellular domain of the human ActRIIA receptor has one or more amino acid substitutions.

[0160] For example, functionally active fragments of the ActRIIA polypeptide can be obtained by screening for polypeptides that recombinantly generate corresponding fragments from nucleic acids encoding the ActRIIA polypeptide. Furthermore, fragments can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid-phase f-Moc or t-Boc chemistry. Fragments can be generated (recombinantly or chemically synthesized) and tested to identify those peptide fragments that can be used as antagonists (inhibitors) of ActRIIA protein or activin-mediated signal transduction.

[0161] Furthermore, for example, functionally active variants of the ActRIIA peptide can be obtained by screening libraries of recombinant modified peptides generated from the corresponding mutagenic nucleic acids encoding the ActRIIA peptide. These variants can be generated and tested to identify those that can be used as antagonists (inhibitors) of ActRIIA protein or activin-mediated signal transduction. In some embodiments, the functional variants of the ActRIIA peptide comprise an amino acid sequence having at least 75% identity with an amino acid sequence selected from SEQ ID NO:2 or 3. In some cases, the functional variants have an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO:2 or 3.

[0162] For example, functional variants of ActRIIA peptides can be generated by modifying their structure for purposes such as enhancing therapeutic efficacy or stability (e.g., ex vivo preservation time or resistance to in vivo proteolytic degradation). When selected to maintain activin binding, such modified ActRIIA peptides can be considered functional equivalents of naturally occurring ActRIIA peptides. Modified ActRIIA peptides can also be generated, for example, through amino acid substitution, deletion, or addition. For instance, there is reason to believe that individual substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of structurally related amino acids (e.g., conserved mutations) will not significantly affect the biological activity of the resulting molecule. Conserved substitutions occur within the amino acid family associated with its side chain. Whether amino acid sequence alterations of ActRIIA peptides produce functional homologs can be readily determined by evaluating the ability of variant ActRIIA peptides to respond in cells in a manner similar to wild-type ActRIIA peptides.

[0163] In some embodiments, the ActRIIA signaling inhibitor used in the compositions and methods provided herein may comprise an ActRIIA polypeptide with one or more specific mutations having altered glycosylation of the polypeptide. Such mutations may introduce or remove one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. The asparagine-linked glycosylation recognition site generally comprises a tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (where “X” is any amino acid), which is specifically recognized by a suitable cellular glycosylation enzyme. Alterations can also be made by adding or substituting one or more serine or threonine residues (for the O-linked glycosylation site) into the sequence of the wild-type ActRIIA polypeptide. Multiple amino acid substitutions or deletions (and / or amino acid deletions at the second position) at one or both of the first or third amino acid positions of the glycosylation recognition site cause deglycosylation at the modified tripeptide sequence. Another way to increase the amount of sugar moiety on the ActRIIA polypeptide is through chemical or enzymatic coupling of the ActRIIA polypeptide with a glycoside. Depending on the coupling method used, the sugar can be linked to (a) arginine and histidine; (b) a free carboxyl group; (c) a free thiol group, such as the free thiol group of cysteine; (d) a free hydroxyl group, such as the free hydroxyl group of serine, threonine, or hydroxyproline; (e) an aromatic residue, such as an aromatic residue of phenylalanine, tyrosine, or tryptophan; or (f) an amide group of glutamine. These methods are described in WO87 / 05330, published September 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated herein by reference. Removal of one or more sugar moieties present on the ActRIIA polypeptide can be achieved chemically or enzymatically. Chemical deglycosylation may involve, for example, exposing the ActRIIA polypeptide to a compound such as trifluoromethanesulfonic acid or an equivalent compound. This treatment causes the cleavage of most or all of the sugars except the linker sugar (N-acetylglucosamine or N-acetylglucosamine), while preserving the integrity of the amino acid sequence. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 2 59:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of the sugar moieties on ActRIIA peptides can be achieved using various endo- and exo-glycosidases, as described by Thotakura et al. (1987) Meth. Enzymol. 138:350.When appropriate, the sequence of the ActRIIA peptide can be adjusted depending on the type of expression system used, as mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns, which may be influenced by the peptide's amino acid sequence. Generally, ActRIIA proteins for humans can be expressed in mammalian cell lines that provide appropriate glycosylation, such as the HEK293 or CHO cell lines, but other expression systems, such as other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells, are also expected to be useful.

[0164] This article further provides methods for generating mutants, particularly combinatorial mutant libraries of ActRIIA peptides, and for truncating mutants; combinatorial mutant libraries are particularly useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries can be to generate ActRIIA peptide variants that can be used, for example, as agonists or antagonists, or as alternatives with novel activities. Several screening assays are provided below, which can be used to evaluate variants. For example, ActRIIA peptide variants can be screened for their ability to bind to ActRIIA ligands, prevent ActRIIA ligands from binding to ActRIIA peptides, or interfere with signal transduction induced by ActRIIA ligands.

[0165] Combinatorial variants with selectively or generally enhanced potency relative to naturally occurring ActRIIA peptides can be generated. Similarly, mutagenesis can produce variants with intracellular half-lives significantly different from the corresponding wild-type ActRIIA peptides. For example, altered proteins can cause greater or less stable responses to proteolytic degradation or other cellular processes that lead to the destruction or inactivation of native ActRIIA peptides. These variants and the genes encoding them can be used to alter ActRIIA peptide levels by modulating the half-lives of the peptides. For example, shorter half-lives can produce more transient biological effects, allowing for tighter control over recombinant ActRIIA peptide levels within the subject. In Fc fusion proteins, mutations can occur at the linker (if present) and / or the Fc moiety to alter the protein's half-lives.

[0166] Combinatorial libraries can be generated by encoding degenerate gene libraries of peptides, each of which includes at least a portion of a possible ActRIIA peptide sequence. For example, a mixture of synthesized oligonucleotides can be enzymatically ligated into gene sequences, such that a degenerate set of possible ActRIIA peptide nucleotide sequences can be expressed as a single peptide, or alternatively as a larger set of fusion proteins (e.g., for phage display).

[0167] There are many methods for generating potential homologous libraries from degenerate oligonucleotide sequences. Degenerate gene sequences can be chemically synthesized in automated DNA synthesizers, and the synthesized genes can then be ligated into suitable vectors for expression. The synthesis of degenerate oligonucleotides is well-known in the art (see, for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). This type of technology has been used in the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and U.S. Patent Nos. 5,223,409, 5,198,346 and 5,096,815).

[0168] Alternatively, other forms of mutagenesis can be used to generate combined libraries. For example, the following methods can be used to generate and isolate ActRIIA peptide variants from a library through screening: e.g., alanine scanning mutagenesis, etc. (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085); mutagenesis by adapter scanning (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); mutagenesis by saturation (Meyers et al., (1986) Science 232:613); mutagenesis by PCR (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or mutagenesis by random mutagenesis, including chemical mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis, especially in combinatorial cases, is an attractive method for identifying truncated (bioactive) forms of ActRIIA peptides.

[0169] Various techniques for screening gene products from combinatorial libraries prepared by point mutation and truncation, and for screening gene products with certain properties in cDNA libraries, are known in the art. These techniques are generally suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of ActRIIA peptides. The most widely used techniques for screening large gene libraries generally involve cloning the gene library into a reproducible expression vector, transforming the resulting vector library into suitable cells, and expressing the combinatorial gene under certain conditions, where the detection of the desired activity facilitates relatively easy isolation of the vector encoding the gene whose product is detected. Preferred assays include activin binding assays and activin-mediated cell signal transduction assays.

[0170] In some embodiments, the ActRIIA peptide used in the inhibitors of the methods and compositions described herein may additionally include post-translational modifications in addition to those naturally present in the ActRIIA peptide. Such modifications may include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Thus, modified ActRIIA peptides may contain non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The role of such non-amino acid elements in the function of the ActRIIA peptide can be tested by any method known to a skilled craftsman. When ActRIIA peptides are generated in cells by cleaving their nascent form, post-translational processing may also be important for the proper folding and / or function of the protein. Different cell types (e.g., CHO, HeLa, MDCK, 293, W138, NIH-3T3, or HEK293) possess specific cellular machinery and unique mechanisms for such post-translational activities and can be selected to ensure the proper modification and processing of ActRIIA peptides.

[0171] In some respects, the functional variants or modified forms of the ActRIIA peptide used in the inhibitors of the methods and compositions described herein include fusion proteins having at least a portion of the ActRIIA peptide and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, multihistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP), or human serum albumin. The fusion domain can be selected to impart desired properties. For example, certain fusion domains are particularly useful for separating fusion proteins by affinity chromatography. For affinity purification, relevant matrices for affinity chromatography are used, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins. Many of these matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress.TM system (Qiagen) for use with (HIS6) fusion couplers. As another example, the fusion domain can be selected to facilitate the detection of ActRIIA peptides. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are typically short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as that of factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In some preferred embodiments, the ActRIIA peptide is fused to a domain of the in vivo stabilized ActRIIA peptide ("stabilizer" domain). "Stabilized" refers to any condition that prolongs serum half-life, whether due to reduced degradation, decreased renal clearance, or other pharmacokinetic effects. Fusion with the Fc portion of immunoglobulins is known to confer desired pharmacokinetic properties on a wide variety of proteins. Similarly, fusion with human serum albumin can confer desired properties. Other types of fusion domains that can be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains (which confer additional biological functions, such as further stimulating bone or muscle growth as needed).

[0172] It is important to understand that the different elements of a fusion protein can be arranged in any way that aligns with the desired function. For example, the ActRIIA peptide can be positioned at the C-terminus of a heterologous domain, or alternatively, the heterologous domain can be positioned at the C-terminus of the ActRIIA peptide. The ActRIIA peptide domain and the heterologous domain are not necessarily adjacent in the fusion protein; other domains or amino acid sequences can be included at the C-terminus or N-terminus of either domain, or between the domains.

[0173] In some embodiments, the ActRIIA peptide used in the inhibitors of the methods and compositions described herein may contain one or more modifications capable of stabilizing the ActRIIA peptide. For example, such modifications may prolong the in vitro half-life of the ActRIIA peptide, prolong the cyclic half-life of the ActRIIA peptide, or reduce the proteolytic degradation of the ActRIIA peptide. Such stabilizing modifications may include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIIA peptide and a stabilizer domain), modifications to glycosylation sites (including, for example, adding glycosylation sites to the ActRIIA peptide), and modifications to the sugar moiety (including, for example, removing the sugar moiety from the ActRIIA peptide). For fusion proteins, the ActRIIA peptide is fused to a stabilizer domain, such as an IgG molecule (e.g., an Fc domain). As used herein, the term "stabilizer domain" refers not only to the fusion domain (e.g., Fc) in the case of fusion proteins, but also includes non-protein modifications such as sugar moieties, or non-protein polymers such as polyethylene glycol.

[0174] In some embodiments, isolated and / or purified forms of ActRIIA peptides, which are derived from or otherwise contain essentially no other proteins, can be used in the methods and compositions described herein. ActRIIA peptides can generally be generated from the expression of recombinant nucleic acids.

[0175] In some respects, the ActRIIA peptides used in the compositions and methods described herein are generated using isolated and / or recombinant nucleic acids encoding any ActRIIA peptide (e.g., soluble ActRIIA peptides), including fragments, functional variants, and fusion proteins disclosed herein. For example, SEQ ID NO:4 encodes a naturally occurring human ActRIIA precursor peptide, while SEQ ID NO:5 encodes a processed extracellular domain of ActRIIA. These nucleic acids can be single-stranded or double-stranded. The nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods for preparing ActRIIA peptides, or as direct therapeutic agents (e.g., in gene therapy methods).

[0176] In some respects, the nucleic acid encoding the ActRIIA polypeptide may include a variant of the nucleic acid SEQ ID NO:4 or 5. Variant nucleotide sequences include sequences differing in one or more nucleotide substitutions, additions, or deletions, such as allelic variants.

[0177] In some embodiments, the isolated or recombinant nucleic acid sequence encoding the ActRIIA polypeptide may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:4 or 5. Those skilled in the art will recognize that nucleic acid sequences complementary to SEQ ID NO:4 or 5, as well as variants of SEQ ID NO:4 or 5, can be used to generate ActRIIA polypeptides suitable for the methods and compositions described herein. In further embodiments, such nucleic acid sequences may be isolated, recombinant, and / or generated from heterologous nucleotide sequences, or derived from a DNA library.

[0178] In other embodiments, the nucleic acid used to generate the ActRIIA polypeptide suitable for the methods and compositions described herein may comprise a nucleotide sequence that hybridizes under high stringency conditions with the nucleotide sequence identified in SEQ ID NO:4 or 5, the complementary sequence of SEQ ID NO:4 or 5, or a fragment thereof. Those skilled in the art will understand that suitable stringency conditions promoting DNA hybridization can be varied. For example, hybridization can be performed at approximately 45°C with 6.0 times sodium chloride / sodium citrate (SSC), followed by washing with 2.0 times SSC at 50°C. For example, the salt concentration in the washing step can be selected from low stringency (approximately 2.0 times SSC at 50°C) to high stringency (approximately 0.2 times SSC at 50°C). Furthermore, the temperature in the washing step can be increased from low stringency conditions at approximately 22°C at room temperature to high stringency conditions at approximately 65°C. Both temperature and salt concentration can be varied, or temperature or salt concentration can remain constant while another variable is changed. In one embodiment, nucleic acid hybridized under low stringency conditions of 6 times SSC at room temperature, followed by washing with 2 times SSC at room temperature, can be used in the methods and compositions described herein.

[0179] Isolated nucleic acids, unlike those listed in SEQ ID NO:4 or 5 due to the degeneracy of the genetic code, can also be used to generate ActRIIA polypeptides suitable for the methods and compositions described herein. For example, multiple amino acids are designated by more than one triplet. Codons or synonymous codons designating the same amino acid (e.g., CAU and CAC are synonymous codons for histidine) can produce “silent” mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that are expected to cause variations in the amino acid sequence of the subject protein can be present in mammalian cells. Those skilled in the art will recognize that such variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acid encoding a particular protein can be present in individuals of a specified species due to natural allelic variations.

[0180] In some embodiments, the recombinant nucleic acid may be efficiently linked to one or more regulatory nucleotide sequences in the expression construct. The regulatory nucleotide sequence will generally be adapted to the host cell used for expression. Many types of suitable expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated herein. Promoters may be naturally occurring promoters or heterozygous promoters combining elements of more than one promoter. The expression construct may be present in a cell or on an episome (e.g., a plasmid), or the expression construct may be inserted into a chromosome. In a preferred embodiment, the expression vector contains a selective marker gene that allows selection of the host cell for transformation. Selective marker genes are well known in the art and will vary depending on the host cell used.

[0181] In some aspects, nucleic acids for generating ActRIIA polypeptides suitable for the methods and compositions described herein may be provided in an expression vector, the expression vector comprising a nucleotide sequence encoding the ActRIIA polypeptide and effectively linked to at least one regulatory sequence. Regulatory sequences are well known and are selected to direct the expression of the ActRIIA polypeptide. Therefore, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For example, any of a variety of expression control sequences that, when effectively linked thereto, control the expression of a DNA sequence may be used in these vectors to express a DNA sequence encoding an ActRIIA polypeptide. Useful expression control sequences include, for example, early and late promoters of SV40, the tet promoter, immediate early promoters of adenovirus or cytomegalovirus, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoters whose expression is directed by T7 RNA polymerase, the major operon and promoter regions of bacteriophage λ, the control region of the ft exosome protein, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases such as Pho5, promoters of yeast α-mating factors, polyhedral promoters of baculovirus systems, and other sequences and various combinations thereof known to control gene expression in prokaryotic and eukaryotic cells or their viruses. It should be understood that the design of expression vectors can depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, the ability to control copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers) should also be considered.

[0182] Recombinant nucleic acids used in the production of ActRIIA peptides suitable for the methods and compositions described herein can be generated by ligating a cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, poultry, insects, or mammals), or both. Expression vectors used for the production of recombinant ActRIIA peptides include plasmids and other vectors. Suitable vectors include, for example, the following plasmid types for expression in prokaryotic cells such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0183] Some mammalian expression vectors contain both a prokaryotic sequence that promotes vector replication in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection into eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids (e.g., pBR322) to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viral derivatives, such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used for transient protein expression in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods used for plasmid preparation and host organism transformation are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination methods, see *Molecular Cloning A Laboratory Manual*, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant peptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., pBlueBac III containing .beta.-gal).

[0184] Vectors can be designed for the production of the subject ActRIIA peptide in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that subject gene constructs can be used to express the subject ActRIIA peptide in cells proliferating in culture to produce proteins, including fusion proteins or variant proteins, for example, for purification.

[0185] Host cells transfected with a recombinant gene including the coding sequence of one or more subject ActRIIA peptides (e.g., SEQ ID NO: 4 or 5) can be used to produce ActRIIA peptides suitable for the methods and compositions described herein. The host cell can be any prokaryotic or eukaryotic cell. For example, the ActRIIA peptides provided herein can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are well known to those skilled in the art.

[0186] Therefore, this article provides a method for producing the ActRIIA peptide. For example, host cells transfected with an expression vector encoding the ActRIIA peptide can be cultured under suitable conditions to induce expression of the ActRIIA peptide. The ActRIIA peptide can be secreted from a mixture of cells and a culture medium containing the ActRIIA peptide, and can be isolated from the mixture. Alternatively, the ActRIIA peptide can be retained in the cytoplasm or membrane portion, harvested from lysed cells, and the isolated protein. Cell cultures include host cells, culture medium, and other byproducts. Suitable culture media for cell culture are well known in the art. Techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific to a particular epitope of the ActRIIA peptide, and affinity purification with reagents binding to the fusion domain of the ActRIIA peptide (e.g., protein A columns can be used to purify ActRIIA-Fc fusions), can be used to isolate the subject ActRIIA peptide from cell culture media, host cells, or both. In a preferred embodiment, the ActRIIA peptide is a fusion protein containing domains that facilitate its purification. In one embodiment, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following in any order: protein A chromatography, Q agarose chromatography, phenyl agarose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished using viral filtration and buffer exchange. As demonstrated herein, the ActRIIA-hFc protein can be purified to >98% purity as determined by size exclusion chromatography and >95% purity as determined by SDS-PAGE. This level of purity is sufficient to achieve the desired effects on the skeleton in mice and to achieve acceptable safety characteristics in mice, rats, and non-human primates.

[0187] In another embodiment, a fusion gene encoding a purified leader sequence, such as a poly-(His) / enterokinase cleavage site sequence, located at the N-terminus of the desired portion of the recombinant ActRIIA polypeptide allows the expressed fusion protein to be purified by affinity chromatography using Ni2+ metalloresin. The purified leader sequence is then removed by treatment with enterokinase to provide the purified ActRIIA polypeptide (see, for example, Hochuli et al., (1987) J. Chromatography 411:177 and Janknecht et al., PNAS USA 88:8972).

[0188] The techniques used to prepare fusion genes are well known. Essentially, following conventional techniques, various DNA fragments encoding different polypeptide sequences are ligated as follows: blunt or staggered ends are used for ligation; restriction endonuclease digestion is performed to provide suitable ends; sticky ends are filled in where appropriate; alkaline phosphatase treatment is used to avoid unwanted ligation; and enzymatic ligation is then performed. In another embodiment, the fusion gene can be synthesized using conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of the gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments, which can then be annealed to produce a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

[0189] The tissue plasminogen leader sequence of SEQ ID NO:9 can be used to express the ActRIIA-Fc fusion protein from the pAID4 vector (SV40 ori / enhancer, CMV promoter) in stably transfected CHO-DUKX Bl 1 cells. The Fc portion is the human IgG1 Fc sequence, as shown in SEQ ID NO:7. In some embodiments, the protein contains approximately 1.5 to 2.5 moles of sialic acid per molecule of ActRIIA-Fc fusion protein at the time of expression.

[0190] In some implementations, the long serum half-life of the ActRIIA-Fc fusion in human subjects can be 25–32 days. Furthermore, the product expressed in CHO cells exhibits a higher affinity for activin B ligands compared to the reported ActRIIA-hFc fusion protein expressed in human 293 cells (del Re et al., J Biol Chem. 2004 Dec 17; 279(51):53126-35). Additionally, while not bound by theory, the use of TPA leader sequences, which provide higher yields than other leader sequences, can provide high-purity N-terminal sequences, unlike ActRIIA-Fc expressed with a natural leader region. The use of natural leader sequences may result in two main types of ActRIIA-Fc, each with a different N-terminal sequence.

[0191] 7.6.2 Inhibitors of ACTRIIB signaling As used herein, the term "ActRIIB" refers to a family of activin type IIB receptor (ActRIIB) proteins from any species and variants derived from such ActRIIB proteins through mutagenesis or other modifications. Reference to ActRIIB herein should be understood as referring to any currently identified form of the receptor. Members of the ActRIIB family are generally transmembrane proteins consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity.

[0192] The ActRIIB signal transduction inhibitors used in the compositions and methods described herein include, but are not limited to, activin-binding soluble ActRIIB peptides; activin-binding peptides (especially activin A or B subunits, also known as β-activin). A or β B Antibodies that bind to ActRIIB; antibodies that bind to ActRIIB and disrupt activin binding; non-antibody proteins that target activin or ActRIIB binding and random peptides that target activin or ActRIIB binding, which may be conjugated to an Fc domain.

[0193] In some embodiments, two or more different proteins (or other portions) having activin or ActRIIB binding activity, particularly activin conjugates that block type I (e.g., soluble type I activin receptor) and type II (e.g., soluble type II activin receptor) binding sites respectively, can be linked together to produce a bifunctional or multifunctional binding molecule that inhibits ActRIIB, and thus can be used in the compositions and methods described herein. In some embodiments, the activin-ActRIIB signaling axis antagonist that inhibits ActRIIB includes nucleic acid aptamers, small molecules, and other agents used in the compositions and methods described herein.

[0194] 7.6.2.1 ActRIIB signal transduction inhibitors containing ActRIIB peptides As used herein, the term "ActRIIB polypeptide" refers to any naturally occurring polypeptide comprising a member of the ActRIIB family, and any variants (including mutants, fragments, fusions, and peptide mimics) that retain their useful activity. For example, ActRIIB polypeptides include polypeptides whose sequences are derived from any known ActRIIB receptor and have at least about 80% identity with the sequence of the ActRIIB polypeptide, optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher. For example, ActRIIB polypeptides can bind to and inhibit the function of ActRIIB proteins and / or activins. Examples of ActRIIB polypeptides include human ActRIIB precursor polypeptides (SEQ ID NO:16 or SEQ ID NO:28). For the ActRIIB precursor polypeptide (i.e., human ActRIIB precursor polypeptide) whose amino acid sequence is described by SEQ ID NO:16 or SEQ ID NO:28, the signal peptide of the ActRIIB precursor polypeptide is located at amino acids 1-18; the extracellular domain is located at amino acids 19-134, and the possible N-glycosylation sites are located at amino acids 42 and 65. SEQ ID NO:19 discloses the nucleic acid sequence encoding the human ActRIIB precursor polypeptide of SEQ ID NO:16 (SEQ ID NO:19 provides alanine at the codon corresponding to amino acid 64, but it can be readily modified by those skilled in the art using methods known in the art to provide arginine instead at the codon corresponding to amino acid 64). See Table 3 for a description of the relevant sequences.

[0195] The amino acid numbers of all ActRIIB-related peptides described herein are based on the amino acid numbers of SEQ ID NO:16 and SEQ ID NO:28 (which differ only in the amino acid expressed at position 64), unless otherwise specified. For example, if an ActRIIB peptide is described as having a substitution / mutation at amino acid position 79, it should be understood that position 79 refers to the 79th amino acid in SEQ ID NO:16 or SEQ ID NO:28 from which the ActRIIB peptide is derived. Similarly, if an ActRIIB peptide is described as having alanine or arginine at amino acid position 64, it should be understood that position 64 refers to the 64th amino acid in SEQ ID NO:16 or SEQ ID NO:28 from which the ActRIIB peptide is derived.

[0196] In some embodiments, the inhibitor of ActRIIB signaling used in the compositions and methods described herein comprises a polypeptide containing an activin-binding domain of ActRIIB. In some embodiments, the activin-binding domain of ActRIIB comprises an extracellular domain of ActRIIB or a portion thereof. In specific embodiments, the extracellular domain of ActRIIB or a portion thereof is soluble. Illustrative modified forms of the ActRIIB polypeptide are disclosed in U.S. Patent Application Publications 20090005308 and 20100068215, the disclosures of which are incorporated herein by reference in their entirety. Illustrative modified forms of the ActRIIB polypeptide are also disclosed in International Patent Application Publications WO 2008 / 097541 and WO 2010 / 019261, the disclosures of which are incorporated herein by reference in their entirety.

[0197] In specific embodiments, the ActRIIB peptides used in the compositions and methods described herein are soluble ActRIIB peptides. The term "soluble ActRIIB peptide" generally refers to a peptide containing the extracellular domain of an ActRIIB protein, including any naturally occurring extracellular domain of the ActRIIB protein and any variants thereof (including mutants, fragments, and peptide mimics). Soluble ActRIIB peptides can bind activin; however, wild-type ActRIIB protein does not exhibit significant selectivity in binding activin to GDF8 / 11. In some embodiments, modified forms of ActRIIB with different binding properties can be used in the methods provided herein. Such modified forms are disclosed, for example, in International Patent Application Publications WO 2006 / 012627 and WO 2010 / 019261, the disclosures of which are incorporated herein by reference in their entirety. Additional specificity for activin can be conjugated to natural or modified ActRIIB proteins by coupling them to a second activin-selective binder. Exemplary soluble ActRIIB peptides include the extracellular domains of human ActRIIB peptides (e.g., SEQ ID NO: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43).

[0198] Fc fusion proteins having the extracellular sequence of ActRIIB disclosed by Hilden et al. (Blood, 1994, 83(8):2163-70), which contain alanine at position 64 (referred to herein as “A64”) corresponding to the ActRIIB precursor amino acid sequence, SEQ ID NO:16, have been shown to have relatively low affinity for activin and GDF-11. In contrast, Fc fusion proteins having arginine at position 64 (referred to herein as “R64”) of the ActRIIB precursor amino acid sequence have affinity for activin and GDF-11 ranging from low nanomolar to high picomolar (see, for example, U.S. Patent Application Publication No. 20100068215, the disclosure of which is incorporated herein in its entirety). See also International Application No. WO 2010 / 019261, the disclosure of which is incorporated herein in its entirety. SEQ ID NO:28 provides the ActRIIB precursor amino acid sequence having arginine at position 64. Therefore, in some embodiments, the ActRIIB polypeptide used according to the compositions and methods described herein may comprise (i) an alanine at position 64 of the amino acid sequence corresponding to the ActRIIB precursor amino acid sequence, i.e., SEQ ID NO:16; or (ii) an arginine at position 64 of the ActRIIB precursor sequence, i.e., SEQ ID NO:28. In other embodiments, the ActRIIB polypeptide used according to the compositions and methods described herein may comprise an amino acid at position 64 of the amino acid sequence corresponding to either SEQ ID NO:16 or SEQ ID NO:28 that is not alanine or arginine.

[0199] It has been shown that the absence of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduces the receptor's affinity for activin (see, for example, Attisano et al., Cell, 1992, 68(1):97-108). The ActRIIB-Fc fusion protein “ActRIIB(20-134)-Fc”, containing amino acids 20-119 (i.e., SEQ ID NO:31) of SEQ ID NO:28, has reduced affinity for GDF-11 and activin compared to the ActRIIB-Fc fusion protein “ActRIIB(20-134)-Fc”, which includes the proline knot region and the intact juxtamembrane domain. However, compared to the untruncated extracellular domain of ActRIIB, the ActRIIB-Fc fusion protein “ActRIIB(20-129)-Fc” containing amino acids 20-129 of SEQ ID NO:28 maintains similar but slightly reduced activity, despite the disruption of its proline knot region. Therefore, ActRIIB peptides containing extracellular domains ending at amino acids 134, 133, 132, 131, 130, and 129 of SEQ ID NO:28 (or SEQ ID NO:16) are expected to be active, but constructs ending at amino acids 134 or 133 may have the highest activity. Similarly, mutations in any of residues 129-134 are not expected to significantly alter ligand-binding affinity, as indicated by the fact that mutations in P129 and P130 of SEQ ID NO:28 do not substantially reduce ligand binding. Therefore, the ActRIIB peptide used according to the methods and compositions described herein may terminate at amino acid 109 (i.e., the last cysteine) as early as SEQ ID NO:28 (or SEQ ID NO:16), however, the ligand binding capacity is expected to be reduced at or between amino acids 109 and 119 of SEQ ID NO:28 (or SEQ ID NO:16).

[0200] Amino acid 29 in SEQ ID NO:16 and SEQ ID NO:28 represents the initial cysteine ​​in the ActRIIB precursor sequence. ActRIIB starting at amino acid 29 at the N-terminus of SEQ ID NO:16 or SEQ ID NO:28, or preceding these amino acid positions, is expected to retain ligand-binding activity. A mutation at position 24 of SEQ ID NO:16 or SEQ ID NO:28, from alanine to asparagine, introduces an N-glycosylation site without substantially affecting ligand binding. This confirms that mutations between the signal cleavage peptide and the cysteine ​​crosslinking region corresponding to amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28 are entirely permissible. Specifically, ActRIIB peptides starting at amino acids 20, 21, 22, 23, and 24 of SEQ ID NO:16 or SEQ ID NO:28 will retain activity, and ActRIIB peptides starting at amino acids 25, 26, 27, 28, and 29 of SEQ ID NO:16 or SEQ ID NO:28 are also expected to retain activity. The ActRIIB polypeptide starting at amino acid positions 22, 23, 24, or 25 of SEQ ID NO:16 or SEQ ID NO:28 may have the greatest activity.

[0201] In summary, the active portion (i.e., ActRIIB polypeptide) of the ActRIIB precursor protein (i.e., SEQ ID NO:16 or SEQ ID NO:28) used according to the methods and compositions described herein generally comprises amino acids 29-109 of SEQ ID NO:16 or SEQ ID NO:28. Such ActRIIB polypeptides may begin, for example, at any residue corresponding to amino acids 19-29 of SEQ ID NO:16 or SEQ ID NO:28, and end at any position corresponding to amino acids 109-134 of SEQ ID NO:16 or SEQ ID NO:28. Specific examples of ActRIIB polypeptides included herein include those beginning at amino acid positions 19-29, 20-29, or 21-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending at amino acid positions 119-134, 119-133, or 129-134, 129-133 of SEQ ID NO:16 or SEQ ID NO:28. Other specific examples of ActRIIB polypeptides included herein include those beginning at amino acid positions 20-24 (or 21-24 or 22-25) of SEQ ID NO:16 or SEQ ID NO:28 and ending at amino acid positions 109-134 (or 109-133), 119-134 (or 119-133), or 129-134 (or 129-133) of SEQ ID NO:16 or SEQ ID NO:28. Variant ActRIIB polypeptides falling within these ranges are also anticipated, particularly those having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or sequence homology with the corresponding portion of SEQ ID NO:16 or SEQ ID NO:28.

[0202] In some embodiments, the inhibitors of ActRIIB signaling used in the compositions and methods described herein comprise a truncated form of the extracellular domain of ActRIIB. The truncation may be at the carboxyl terminus and / or amino terminus of the ActRIIB polypeptide. In some embodiments, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths relative to the mature ActRIIB polypeptide extracellular domain. In some embodiments, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 N-terminal amino acids of the mature ActRIIB polypeptide extracellular domain. In some embodiments, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the extracellular domain of a mature ActRIIB polypeptide. For example, truncated forms of ActRIIB include polypeptides having amino acids 20-119; 20-128; 20-129; 20-130; 20-131; 20-132; 20-133; 20-134; 20-131; 21-131; 22-131; 23-131; 24-131, and 25-131, wherein the amino acid positions refer to the amino acid positions in SEQ ID NO:16 or SEQ ID NO:28.

[0203] Other exemplary truncated forms of ActRIIB include (i) a polypeptide beginning with any one of amino acids 21-29 of SEQ ID NO:16 or SEQ ID NO:28 (optionally beginning with 21-25 of SEQ ID NO:16 or SEQ ID NO:28) and ending with any one of amino acids 109-134 of SEQ ID NO:16 or SEQ ID NO:28; (ii) a polypeptide beginning with any one of amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28 (optionally beginning with 20-25 of SEQ ID NO:16 or SEQ ID NO:28) and ending with any one of amino acids 109-133 of SEQ ID NO:16 or SEQ ID NO:28; (iii) a polypeptide beginning with any one of amino acids 20-24 of SEQ ID NO:16 or SEQ ID NO:28 (optionally beginning with 20-25 of SEQ ID NO:16 or SEQ ID NO:28) and ending with any one of amino acids 109-133 of SEQ ID NO:16 or SEQ ID NO:28. (iv) A polypeptide starting with any one of amino acids 109-133 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 109-134 of SEQ ID NO:16 or SEQ ID NO:28; (v) A polypeptide starting with any one of amino acids 20-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 118-133 of SEQ ID NO:16 or SEQ ID NO:28; (vi) A polypeptide starting with any one of amino acids 21-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 118-134 of SEQ ID NO:16 or SEQ ID NO:28; (vii) A polypeptide starting with any one of amino acids 20-24 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 118-134 of SEQ ID NO:16 or SEQ ID NO:28. (viii) A polypeptide that begins with any one of amino acids 128-133 of SEQ ID NO:28 or SEQ ID NO:28 and ends with any one of amino acids 128-133 of SEQ ID NO:16 or SEQ ID NO:28; (ix) A polypeptide that begins with any one of amino acids 21-29 of SEQ ID NO:16 or SEQ ID NO:28 and ends with any one of amino acids 118-134 of SEQ ID NO:16 or SEQ ID NO:28;(x) a polypeptide beginning with any one of amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 118-133 of SEQ ID NO:16 or SEQ ID NO:28; (xi) a polypeptide beginning with any one of amino acids 21-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 128-134 of SEQ ID NO:16 or SEQ ID NO:28; and (xii) a polypeptide beginning with any one of amino acids 20-29 of SEQ ID NO:16 or SEQ ID NO:28 and ending with any one of amino acids 128-133 of SEQ ID NO:16 or SEQ ID NO:28. In one specific embodiment, the ActRIIB polypeptide comprises, is substantially composed of, or is composed of the amino acid sequence beginning with amino acid position 25 of SEQ ID NO:16 or SEQ ID NO:28 and ending with amino acid position 131 of SEQ ID NO:16 or SEQ ID NO:28. In another specific embodiment, ActRIIB consists of, or substantially consists of, the amino acid sequence of SEQ ID NO: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, or 43.

[0204] Any of the ActRIIB peptides used in the compositions and methods described herein can be produced as homodimers. Any of the ActRIIB peptides used in the compositions and methods described herein can be formulated into fusion proteins having a heterologous moiety comprising a constant region from the IgG heavy chain, such as an Fc domain. Any of the ActRIIB peptides used in the compositions and methods described herein may contain an acidic amino acid at position 79 corresponding to SEQ ID NO:16 or SEQ ID NO:28, optionally substituted, deleted, or inserted with one or more other amino acids relative to SEQ ID NO:16 or SEQ ID NO:28.

[0205] In specific embodiments, the inhibitors of ActRIIB signaling used in the compositions and methods described herein comprise the extracellular domain of ActRIIB having one or more amino acid substitutions / mutations. Such amino acid substitutions / mutations can be, for example, exchanging leucine at position 79 of amino acid SEQ ID NO:16 or SEQ ID NO:28 for an acidic amino acid, such as aspartic acid or glutamate. For example, position L79 of SEQ ID NO:16 or SEQ ID NO:28 in the ActRIIB extracellular domain polypeptide can be altered to confer altered activin-myosostatin (GDF-11) binding properties. The L79A and L79P mutations significantly reduce GDF-11 binding compared to activin binding. The L79E and L79D mutations maintain GDF-11 binding while indicating a substantial reduction in activin binding.

[0206] In some embodiments, the ActRIIB signaling inhibitor used in the compositions and methods described herein comprises a truncated form of the ActRIIB extracellular domain, which also has an amino acid substitution, such as the exchange of amino acid position 79 of SEQ ID NO:16 or SEQ ID NO:28 with an acidic amino acid, such as aspartic acid or glutamic acid. In a specific embodiment, the truncated form of the extracellular domain of the ActRIIB polypeptide also having an amino acid substitution used in the compositions and methods described herein is SEQ ID NO:23. The truncated and / or ActRIIB form with one or more amino acid substitutions may be linked to the Fc domain of the antibody discussed above.

[0207] For example, functionally active fragments of the ActRIIB peptide can be obtained by screening for peptides that recombinantly generate corresponding fragments of nucleic acids encoding the ActRIIB peptide. Furthermore, fragments can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid-phase f-Moc or t-Boc chemistry. Fragments can be generated (recombinantly or chemically synthesized) and tested to identify those peptide fragments that can be used as antagonists (inhibitors) of ActRIIB protein or activin-mediated signal transduction.

[0208] Furthermore, variants of the ActRIIB peptide with functional activity can be obtained, for example, by screening libraries of recombinant modified peptides generated from corresponding mutagenic nucleic acids encoding the ActRIIB peptide. These variants can be generated and tested to identify those that can be used as antagonists (inhibitors) of ActRIIB protein or activin-mediated signal transduction. In some embodiments, the functional variants of the ActRIIB peptide include amino acid sequences having at least 75% identity with the amino acid sequences selected from SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43. In some embodiments, the functional variants have amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences selected from SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43.

[0209] Functional variants of ActRIIB peptides can be generated, for example, by modifying their structure, for purposes such as improving therapeutic efficacy or stability (e.g., ex vivo preservation time or resistance to in vivo proteolytic degradation). When selected to maintain activin binding, such modified ActRIIB peptides can be considered functional equivalents of naturally occurring ActRIIB peptides. Modified ActRIIB peptides can also be generated through, for example, amino acid substitution, deletion, or addition. For example, there is reason to believe that replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine alone, or similar substitutions of amino acids with structurally related amino acids (e.g., conserved mutations) will not significantly affect the biological activity of the resulting molecule. Conserved substitutions occur within the amino acid family associated with its side chain. Whether amino acid sequence alterations of ActRIIB peptides produce functional homologs can be readily determined by evaluating the ability of variant ActRIIB peptides to respond in cells in a manner similar to that of wild-type ActRIIB peptides.

[0210] ActRIIB peptide mutants, particularly collections of combined mutants of ActRIIB peptides and truncated mutants; libraries of combined mutants are particularly useful for identifying functional variant sequences that can be used in the methods and compositions described herein. The purpose of screening such libraries may be to generate, for example, ActRIIB peptide variants that can be used as agonists or antagonists, or alternatives, which together possess novel activities.

[0211] It has been shown that the ligand-binding bag of ActRIIB is defined by residues Y31, N33, N35, L38 to T41, E47, E50, Q53 to K55, L57, H58, Y60, S62, K74, W78 to N83, Y85, R87, A92, and E94 to F101 in SEQ ID NO:16 or SEQ ID NO:28. At these positions, conserved mutations are expected to be permissible, but the K74A mutation is fully permissible, as are mutations at R40A, K55A, F82A, and L70. In the African toad (Xenopus), R40 is K, indicating that basic amino acids at this position are permissible. In bovine ActRIIB, Q53 is R, and in African toad ActRIIB, it is K; therefore, amino acids including R, K, Q, N, and H are permissible at this position. Therefore, the general formula for ActRIIB peptides used in the methods and compositions described herein is to contain amino acids 29-109 of SEQ ID NO:16 or SEQ ID NO:28, but optionally starting at amino acid positions 20-24 or 22-25 of SEQ ID NO:16 or SEQ ID NO:28 and ending at amino acid positions ranging from 129-134 of SEQ ID NO:16 or SEQ ID NO:28, and containing no more than 1, 2, 5, or 15 conserved amino acid changes in the ligand-binding bag, and zero or one or more non-conserved changes at positions 40, 53, 55, 74, 79, and / or 82 of amino acids of SEQ ID NO:16 or SEQ ID NO:28 in the ligand-binding bag. Such ActRIIB peptides may maintain greater than 80%, 90%, 95%, or 99% sequence identity or sequence homology with the amino acid sequence 29-109 of SEQ ID NO:16 or SEQ ID NO:28. Sites outside the binding bag, where variability is particularly permissible, include the amino and carboxyl termini of the extracellular domain of ActRIIB and positions 42-46 and 65-73. The change from asparagine to alanine at position 65 in SEQ ID NO:16 or SEQ ID NO:28 (N65A) actually improves ligand binding in the A64 background, and therefore no detrimental effect on ligand binding is expected in the R64 background. This change may remove the glycosylation at N65 in the A64 background, thus indicating that significant changes in this region are likely permissible. While the R64A change is less permissible, R64K is completely permissible, therefore another basic residue, such as H at position 64, is permissible.

[0212] As a specific example of an ActRIIB peptide with a mutation in its ligand-binding domain, the positively charged amino acid residue Asp (D80) of the ligand-binding domain of ActRIIB can be mutated to a different amino acid residue, causing the variant ActRIIB peptide to preferentially bind GDF8 instead of activin. In one specific embodiment, the D80 residue is replaced with an amino acid residue selected from: uncharged amino acid residues, negatively charged amino acid residues, and hydrophobic amino acid residues. As a more specific example, the hydrophobic residue L79 can be replaced with the acidic amino acid aspartic acid or glutamic acid to significantly reduce activin binding while maintaining GDF11 binding. As is recognized by those skilled in the art, most of the aforementioned mutations, variations, or modifications can be achieved at the nucleic acid level, or in some cases, through post-translational modification or chemical synthesis. Such techniques are well known in the art.

[0213] In specific embodiments, the inhibitors of ActRIIB signaling used in the compositions and methods described herein comprise conjugates / fusion proteins containing an extracellular domain (e.g., an activin-binding domain) of the ActRIIB receptor linked to the Fc portion of an antibody. Such conjugates / fusion proteins may comprise any ActRIIB polypeptide disclosed herein (e.g., any one of SEQ ID NO: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, or 43), any ActRIIB polypeptide known in the art, or any ActRIIB polypeptide generated using methods known in the art and / or provided herein.

[0214] In some embodiments, the extracellular domain is linked to the Fc moiety of the antibody via a linker (e.g., a peptide linker). Exemplary linkers comprise short polypeptide sequences, such as 2–10, 2–5, 2–4, or 2–3 amino acid residues (e.g., glycine residues), for example, a Gly-Gly-Gly linker. In one specific embodiment, the linker comprises the amino acid sequence Gly-Gly-Gly (GGG). In another specific embodiment, the linker comprises the amino acid sequence Thr-Gly-Gly-Gly (TGGG). Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In some cases, the mutant Fc domain having one or more of these mutations (e.g., the Asp-265 mutation) has a reduced ability to bind to the Fcγ receptor compared to the wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., the Asn-434 mutation) has an increased ability to bind to the MHC class I-associated Fc receptor (FcRN) compared to the wild-type Fc domain. Exemplary fusion proteins containing the soluble extracellular domain of ActRIIB fused with the Fc domain are listed in SEQ ID NO: 20, 21, 24, 25, 34, 35, 38, 39, 40, 41, 44, 46 and 47.

[0215] In one specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein comprises an extracellular domain or a portion thereof of an ActRIIB linked to the Fc portion of an antibody, wherein the ActRIIB signaling inhibitor comprises an amino acid sequence having at least 75% identity with an amino acid sequence selected from SEQ ID NO: 20, 21, 24, 25, 34, 35, 38, 39, 40, 41, 44, 46, and 47. In another specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein comprises an extracellular domain or a portion thereof of an ActRIIB linked to the Fc portion of an antibody, wherein the ActRIIB signaling inhibitor comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 20, 21, 24, 25, 34, 35, 38, 39, 40, 41, 44, 46, and 47.

[0216] In one specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein is a fusion protein between the extracellular domain of the human ActRIIB receptor and the Fc moiety of IgG1. In another specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein is a fusion protein between a truncated extracellular domain of the human ActRIIB receptor and the Fc moiety of IgG1. In yet another specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein is a fusion protein between a truncated extracellular domain of the human ActRIIB receptor and the Fc moiety of IgG1, wherein the truncated extracellular domain of the human ActRIIB receptor has an amino acid substitution at amino acid position 79 corresponding to amino acid SEQ ID NO:16 or SEQ ID NO:28. In one embodiment, the amino acid substitution at amino acid position 79 corresponding to amino acid SEQ ID NO:16 or SEQ ID NO:28 is a leucine substitution with aspartic acid (i.e., an L79D mutation).

[0217] In one specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein is SEQ ID NO:24 or 25, which represents a fusion protein between the extracellular domain of the human ActRIIB receptor and the Fc portion of IgG1, wherein the ActRIIB extracellular domain comprises amino acids 25-131 of SEQ ID NO:28 with an L79D mutation. SEQ ID NO:45 lists the nucleic acid sequence encoding the ActRIIB-Fc fusion protein of SEQ ID NO:24.

[0218] In another specific embodiment, the ActRIIB signaling inhibitor used in the compositions and methods described herein is SEQ ID NO:34 or 35, which represents a fusion protein between the extracellular domain of the human ActRIIB receptor and the Fc portion of IgG1, wherein the extracellular domain of the ActRIIB contains amino acids 25-131 of SEQ ID NO:16 with the L79D mutation.

[0219] The asparagine-linked glycosylation recognition site typically comprises a tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (where "X" is any amino acid), which is specifically recognized by a suitable cellular glycosylation enzyme. It can also be altered by adding or substituting one or more serine or threonine residues (for the O-linked glycosylation site) into the sequence of the wild-type ActRIIB peptide. Substitution or deletion of one or both amino acids at the first or third amino acid position of the glycosylation recognition site (and / or deletion of an amino acid at the second position) causes deglycosylation at the modified tripeptide sequence. Another way to increase the amount of sugar moiety on the ActRIIB peptide is through chemical or enzymatic coupling of glycosides to the ActRIIB peptide. Depending on the coupling method used, the sugar can be linked to (a) arginine and histidine; (b) a free carboxyl group; (c) a free thiol group, such as the free thiol group of cysteine; (d) a free hydroxyl group, such as the hydroxyl group of serine, threonine, or hydroxyproline; (e) an aromatic residue, such as an aromatic residue of phenylalanine, tyrosine, or tryptophan; or (f) an amide group of glutamine. These methods are described in WO 87 / 05330, published September 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated herein by reference. Removal of one or more sugar moieties present on the ActRIIB polypeptide can be achieved chemically or enzymatically. Chemical deglycosylation may involve, for example, exposing the ActRIIB polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment causes the cleavage of most or all of the sugars except the linker sugar (N-acetylglucosamine or N-acetylglucosamine), while preserving the integrity of the amino acid sequence. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of the sugar moiety on ActRIIB peptides can be achieved using various endo- and exo-glycosidases, as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. Depending on the type of expression system used, the sequence of the ActRIIB peptide can be modified as appropriate, as mammalian, yeast, insect, and plant cells may all introduce different glycosylation patterns that may be influenced by the amino acid sequence of the peptide. Generally, ActRIIB proteins for human use can be expressed in mammalian cell lines that provide appropriate glycosylation, such as HEK293 or CHO cell lines, but other expression systems, such as other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells, are also expected to be useful.

[0220] In specific embodiments, ActRIIB peptides containing mutations with more N-glycosylation sites (NXS / T) that extend the serum half-life of the ActRIIB-Fc fusion protein, relative to the ActRIIB(R64)-Fc form, can be used in the methods and compositions described herein. In one specific embodiment, asparagine (A24N) is introduced at position 24 of SEQ ID NO:16 or SEQ ID NO:28 to generate an NXT sequence conferring a longer half-life. Other NX(T / S) sequences may be present at positions 42-44 (NQS) and 65-67 (NSS), but the latter cannot be effectively glycosylated with the R at position 64 (i.e., in the R64 peptide). Generally, NXS / T sequences can be introduced outside the ligand-binding pocket of ActRIIB, as described in detail above. Particularly suitable sites for introducing non-endogenous NXS / T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134, or 129-134 of SEQ ID NO:16 or SEQ ID NO:28. NXS / T sequences can also be introduced into linkers between ActRIIB sequences and Fc or other fusion components. Such sites can be introduced with minimal effort by introducing N at the correct position relative to a pre-existing S or T, or by introducing S or T at a position corresponding to a pre-existing N. Therefore, desirable alterations for generating N-glycosylation sites are: A24N, R64N, S67N (possibly in combination with the N65A alteration), E106N, R112N, G120N, E123N, P129N, A132N, R112S, and R112T (all amino acid positions correspond to their positions in SEQ ID NO:16 or SEQ ID NO:28). It is anticipated that any S to be glycosylated can be changed to T, since glycosylation provides protection without creating an immunogenic site. Similarly, it is anticipated that any T to be glycosylated can be changed to S. Therefore, this paper includes S67T and S44T variations. Likewise, in the A24N variant, the S26T alteration can be used. Thus, the ActRIIB peptide may include one or more additional non-endogenous N-glycosylation concordant sequences.

[0221] Various screening assays can be used to evaluate ActRIIB peptide variants. For example, ActRIIB peptide variants can be screened for their ability to bind to ActRIIB ligands, prevent ActRIIB ligands from binding to ActRIIB peptides, or interfere with signal transduction induced by ActRIIB ligands. The activity of ActRIIB peptides or their variants can also be tested in cell-based or in vivo assays.

[0222] Combinatorially derived variants can be generated, exhibiting selectively or generally enhanced potency relative to naturally occurring ActRIIB peptides. Similarly, mutagenesis can result in variants with intracellular half-lives significantly different from the corresponding wild-type ActRIIB peptides. For example, altered proteins can be conferred as more or less stable to proteolytic degradation or other cellular metabolic processes that cause destruction or inactivation of native ActRIIB peptides. ActRIIB peptide levels can be altered by utilizing these variants and the genes encoding them, through modulation of the half-lives of ActRIIB peptides. For example, shorter half-lives can produce shorter biological effects, allowing for tighter control over recombinant ActRIIB peptide levels in the body. In Fc fusion proteins, mutations can occur at the linker (if present) and / or the Fc moiety to alter the protein's half-lives.

[0223] Combinatorial libraries can be generated by encoding degenerate gene libraries of peptides, each of which includes at least a portion of a possible ActRIIB peptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically linked to gene sequences, so that a degenerate set of possible ActRIIB peptide nucleotide sequences can be expressed as a single peptide, or alternatively, as a larger set of fusion proteins (e.g., for phage display).

[0224] Numerous methods exist for generating libraries of potential homologues from degenerate oligonucleotide sequences. Degenerate gene sequences can be chemically synthesized in automated DNA synthesizers, and the synthesized genes can then be ligated into suitable vectors for expression. The synthesis of degenerate oligonucleotides is well-known in the art (see, for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). This type of technology has been applied to the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and U.S. Patent Nos. 5,223,409, 5,198,346 and 5,096,815).

[0225] Alternatively, other forms of mutagenesis can be used to generate combined libraries. For example, the following methods can be used to generate and isolate ActRIIB peptide variants from a library through screening: for example, alanine scanning mutagenesis, etc. (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085); mutagenesis by adapter scanning (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); mutagenesis by saturation (Meyers et al., (1986) Science 232:613); mutagenesis by PCR (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or mutagenesis by random mutagenesis, including chemical mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis, especially in combinatorial cases, is an attractive method for identifying truncated (bioactive) forms of ActRIIB peptides.

[0226] Various techniques for screening gene products from combinatorial libraries prepared by point mutation and truncation, and for screening gene products with certain properties in cDNA libraries, are known in the art. These techniques are generally suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of ActRIIB peptides. The most widely used techniques for screening large gene libraries generally involve cloning the gene library into a reproducible expression vector, transforming the resulting vector library into suitable cells, and expressing the combinatorial gene under certain conditions, where the detection of the desired activity facilitates relatively easy isolation of the vector encoding the gene whose product is detected. Preferred assays include activin binding assays and activin-mediated cell signal transduction assays.

[0227] In some embodiments, the ActRIIB peptide used in the inhibitors of the methods and compositions described herein may further comprise post-translational modifications in addition to those naturally present in the ActRIIB peptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Thus, the modified ActRIIB peptide may contain non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides, or monosaccharides and phosphates. The role of such non-amino acid elements in the function of the ActRIIB peptide can be tested by any method known to a skilled craftsman. When the ActRIIB peptide is generated in cells by cleaving the nascent form of the ActRIIB peptide, post-translational processing may also be important for the proper folding and / or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, W138, NIH-3T3, or HEK293) possess specific cellular machinery and unique mechanisms for this post-translational activity, which can be selected to ensure the proper modification and processing of the ActRIIB peptide.

[0228] In some respects, functional variants or modified forms of ActRIIB peptides include fusion proteins having at least a portion of the ActRIIB peptide and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, multihistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP), or human serum albumin. Fusion domains can be selected to impart desired properties. For example, certain fusion domains are particularly useful for separating fusion proteins by affinity chromatography. For affinity purification, affinity chromatography-specific matrices are used, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress.TM system (Qiagen) for use with (HIS6) fusion couplers. As another example, fusion domains can be selected to facilitate the detection of ActRIIB peptides. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are typically short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as that of factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In some preferred embodiments, the ActRIIB peptide is fused to a domain of the in vivo stabilized ActRIIB peptide ("stabilizer" domain). "Stabilized" refers to any condition that prolongs serum half-life, whether due to reduced degradation, decreased renal clearance, or other pharmacokinetic effects. Fusion with the Fc portion of immunoglobulins is known to confer desired pharmacokinetic properties on a wide variety of proteins. Similarly, fusion with human serum albumin can confer desired properties. Other types of fusion domains that can be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains (which confer additional biological functions, such as further stimulating bone or muscle growth as needed).

[0229] It is important to understand that the different elements of a fusion protein can be arranged in any way that aligns with the desired function. For example, the ActRIIB peptide can be positioned at the C-terminus of a heterologous domain, or alternatively, the heterologous domain can be positioned at the C-terminus of the ActRIIB peptide. The ActRIIB peptide domain and the heterologous domain are not necessarily adjacent in the fusion protein; other domains or amino acid sequences can be included at the C-terminus or N-terminus of either domain, or between the domains.

[0230] In some embodiments, the ActRIIB peptide used in the methods and compositions described herein contains one or more modifications capable of stabilizing the ActRIIB peptide. For example, such modifications can prolong the in vitro half-life of the ActRIIB peptide, prolong the cyclic half-life of the ActRIIB peptide, or reduce the proteolytic degradation of the ActRIIB peptide. Such stabilizing modifications can include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIIB peptide and a stabilizer domain), modifications to glycosylation sites (including, for example, adding glycosylation sites to the ActRIIB peptide), and modifications to the sugar moiety (including, for example, removing the sugar moiety from the ActRIIB peptide). For fusion proteins, the ActRIIB peptide is fused to a stabilizer domain, such as an IgG molecule (e.g., an Fc domain). As used herein, the term "stabilizer domain" refers not only to the fusion domain (e.g., Fc) in the case of fusion proteins, but also includes non-protein modifications such as sugar moieties, or non-protein polymers such as polyethylene glycol.

[0231] In some embodiments, the methods and compositions described herein utilize isolated and / or purified ActRIIB peptides, i.e., ActRIIB peptides isolated from or otherwise substantially free of other proteins, which can be used in the methods and compositions described herein. ActRIIB peptides can generally be generated by expression from recombinant nucleic acids.

[0232] In some respects, the ActRIIB peptides used in the methods and compositions described herein are encoded by isolated and / or recombinant nucleic acids, including fragments, functional variants, and fusion proteins disclosed herein. For example, SEQ ID NO:19 encodes a naturally occurring human ActRIIB precursor peptide. The subject nucleic acid can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods for preparing ActRIIB peptides, or as direct therapeutic agents (e.g., in gene therapy methods).

[0233] In some respects, to further understand, nucleic acids that can be used to generate ActRIIB polypeptides suitable for the methods and compositions described herein include nucleic acids as variants of SEQ ID NO:19, as well as variants of those nucleic acid sequences encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43). Variant nucleotide sequences include sequences differing in one or more nucleotide substitutions, additions, or deletions, such as allelic variants.

[0234] In some embodiments, the isolated or recombinant nucleic acid sequence used to generate the ActRIIB polypeptide suitable for the methods and compositions described herein has at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:19 or those nucleic acid sequences encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43). Those skilled in the art will recognize that nucleic acid sequences complementary to SEQ ID NO:19 or those encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43), as well as variants of SEQ ID NO:19 or those encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43), can be used in the methods and compositions described herein. In a further embodiment, the nucleic acid sequence may be isolated, recombinant, and / or fused with a heterologous nucleotide sequence, or may be in a DNA library.

[0235] In other embodiments, the nucleic acids used to generate ActRIIB polypeptides suitable for the methods and compositions described herein include nucleotide sequences that hybridize under highly stringent conditions with the nucleotide sequences specified in SEQ ID NO:19 or those encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43), the complementary sequences of SEQ ID NO:19, or fragments thereof encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43). Those skilled in the art will recognize that suitable stringent conditions promoting DNA hybridization can be modified. For example, hybridization can be performed at 6.0 times sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by washing with 2.0 times SSC at 50°C. For example, the salt concentration in the washing step can be selected from low stringency (approximately 2.0 times the SSC concentration at 50°C) to high stringency (approximately 0.2 times the SSC concentration at 50°C). Furthermore, the temperature in the washing step can be increased from low stringency conditions at room temperature (approximately 22°C) to high stringency conditions (approximately 65°C). Both temperature and salt concentration can be varied, or either temperature or salt concentration can remain constant while the other variable changes. In one embodiment, nucleic acids hybridized under low stringency conditions of 6 times the SSC concentration at room temperature, followed by washing with 2 times the SSC concentration at room temperature, can be used in the methods and compositions described herein.

[0236] Isolated nucleic acids, due to the degeneracy of the genetic code, that differ from those nucleic acids listed in SEQ ID NO:19 or those encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO:17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43), can also be used to generate ActRIIB polypeptides suitable for the methods and compositions described herein. For example, many amino acids are designated by more than one triplet. Codons designating the same amino acid, or synonymous codons (e.g., CAU and CAC are synonymous for histidine), can produce “silent” mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that are expected to cause changes in the amino acid sequence of the subject protein can exist in mammalian cells. Those skilled in the art will recognize that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acid encoding a particular protein can exist in individuals of a specified species due to natural allelic variations. Any and all such nucleotide variations and resulting amino acid polymorphisms can be used in the methods and compositions described herein.

[0237] In some embodiments, the recombinant nucleic acid may be efficiently linked to one or more regulatory nucleotide sequences in the expression construct. The regulatory nucleotide sequence will generally be adapted to the host cell used for expression. Many types of suitable expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art can be used in the methods and compositions described herein. The promoter may be a naturally occurring promoter or a heterozygous promoter combining elements of more than one promoter. The expression construct may be present in a cell or on an episome (e.g., a plasmid), or the expression construct may be inserted into a chromosome. In a preferred embodiment, the expression vector contains a selective marker gene, allowing selection of the transformed host cell. Selective marker genes are well known in the art and will vary depending on the host cell used.

[0238] In some aspects, nucleic acids in expression vectors are provided for the production of ActRIIB polypeptides suitable for the methods and compositions described herein, said expression vectors comprising nucleotide sequences encoding the ActRIIB polypeptide and effectively linked to at least one regulatory sequence. Regulatory sequences are well-known and are selected to direct the expression of the ActRIIB polypeptide. Therefore, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For example, any of a variety of expression control sequences that, when effectively linked thereto, control the expression of a DNA sequence can be used in these vectors to express a DNA sequence encoding an ActRIIB polypeptide. Useful expression control sequences include, for example, early and late promoters of SV40, the tet promoter, immediate early promoters of adenovirus or cytomegalovirus, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoters whose expression is directed by T7 RNA polymerase, the major operon and promoter regions of bacteriophage λ, the control region of the ft exosome protein, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases such as Pho5, promoters of yeast α-mating factors, polyhedral promoters of baculovirus systems, and other sequences and various combinations thereof known to control gene expression in prokaryotic and eukaryotic cells or their viruses. It should be understood that the design of expression vectors can depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, the ability to control copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers) should also be considered.

[0239] Recombinant nucleic acids can be produced by ligating a cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vectors used for the production of recombinant ActRIIB peptides include plasmids and other vectors. Suitable vectors include, for example, the following plasmid types for expression in prokaryotic cells such as Escherichia coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0240] Some mammalian expression vectors contain both a prokaryotic sequence that promotes vector replication in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection into eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids (e.g., pBR322) to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viral derivatives, such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used for transient protein expression in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods used for plasmid preparation and host organism transformation are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination methods, see *Molecular Cloning A Laboratory Manual*, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant peptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., pBlueBac III containing .beta.-gal).

[0241] In one embodiment, the vector may be designed for the production of the ActRIIB polypeptide used in the methods and compositions described herein in CHO cells, for example, the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that the subject gene construct can be used to express the subject ActRIIB polypeptide in cells proliferating in a culture to produce, for example, proteins for purification, including fusion proteins or variant proteins.

[0242] Host cells transfected with a recombinant gene comprising a coding sequence of one or more subject ActRIIB polypeptides (e.g., SEQ ID NO: 19) or those nucleic acid sequences encoding soluble ActRIIB polypeptides (e.g., nucleic acids encoding SEQ ID NO: 17, 18, 23, 26, 27, 29, 30, 31, 32, 33, 36, 37, 42, and 43) can be used to produce ActRIIB polypeptides suitable for the methods and compositions described herein. The host cell can be any prokaryotic or eukaryotic cell. For example, the ActRIIB polypeptide can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are well known to those skilled in the art.

[0243] Therefore, this document provides a method for producing the ActRIIB peptide used in the methods and compositions described herein. For example, host cells transfected with an expression vector encoding the ActRIIB peptide can be cultured under suitable conditions to induce expression of the ActRIIB peptide. The ActRIIB peptide can be secreted from a mixture of cells and a culture medium containing the ActRIIB peptide, and can be isolated from the mixture. Alternatively, the ActRIIB peptide can be retained in the cytoplasm or membrane portion, harvested as lysed cells, and the isolated protein. Cell cultures include host cells, culture medium, and other byproducts. Suitable culture media for cell culture are well known in the art. Techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific to a particular epitope of the ActRIIB peptide, and affinity purification with reagents binding to the fusion domain of the ActRIIB peptide (e.g., protein A columns can be used to purify ActRIIB-Fc fusions), can be used to isolate the subject ActRIIB peptide from the cell culture medium, host cells, or both. In a preferred embodiment, the ActRIIB peptide is a fusion protein containing domains that facilitate its purification. In one embodiment, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following in any order: protein A chromatography, Q agarose chromatography, phenyl agarose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished using viral filtration and buffer exchange. As demonstrated herein, the ActRIIB-hFc protein can be purified to >98% purity as determined by size exclusion chromatography and >95% purity as determined by SDS-PAGE. This level of purity is sufficient to achieve the desired effects on the skeleton in mice and to achieve acceptable safety characteristics in mice, rats, and non-human primates.

[0244] In another embodiment, a fusion gene encoding a purified leader sequence, such as a poly-(His) / enterokinase cleavage site sequence, located at the N-terminus of the desired portion of the recombinant ActRIIB polypeptide can allow the expressed fusion protein to be purified by affinity chromatography using Ni2+ metalloresin. The purified leader sequence is then removed by treatment with enterokinase to provide the purified ActRIIB polypeptide (see, for example, Hochuli et al., (1987) J. Chromatography 411:177 and Janknecht et al., PNAS USA 88:8972).

[0245] The techniques used to prepare fusion genes are well known. Essentially, following conventional techniques, various DNA fragments encoding different polypeptide sequences are ligated as follows: blunt or staggered ends are used for ligation; restriction endonuclease digestion is performed to provide suitable ends; sticky ends are filled in where appropriate; alkaline phosphatase treatment is used to avoid unwanted ligation; and enzymatic ligation is then performed. In another embodiment, the fusion gene can be synthesized using conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of the gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments, which can then be annealed to produce a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

[0246] The tissue plasminogen leader sequence of SEQ ID NO:8 can be used to express the ActRIIB-Fc fusion protein from the pAID4 vector (SV40 ori / enhancer, CMV promoter) in stably transfected CHO-DUKX Bl 1 cells. The Fc portion may contain a human IgG1 Fc sequence, as shown in SEQ ID NO:7. In some embodiments, the protein contains approximately 1.5 to 2.5 moles of sialic acid per molecule of ActRIIB-Fc fusion protein at the time of expression.

[0247] In some implementations, the long serum half-life of the ActRIIB-Fc fusion in human subjects can be 25–32 days. Furthermore, the product expressed in CHO cells exhibits a higher affinity for activin B ligands compared to the reported ActRIIB-hFc fusion protein expressed in human 293 cells (del Re et al., J Biol Chem. 2004 Dec 17; 279(51):53126-35). Additionally, while not bound by theory, the use of TPA leader sequences, which provide higher yields than other leader sequences, can provide high-purity N-terminal sequences, unlike ActRIIB-Fc expressed with a natural leader region. The use of natural leader sequences may result in two main types of ActRIIB-Fc, each with a different N-terminal sequence.

[0248] 7.6.3 Other ActRII receptor signal transduction inhibitors In some embodiments, the ACTRII signal transduction inhibitor used in the compositions and methods described herein is a nucleic acid compound.

[0249] Examples of nucleic acid compounds that inhibit the ActRII receptor include antisense nucleic acids, siRNA or RNAi constructs, and catalytic nucleic acid constructs. Nucleic acid compounds can be single-stranded or double-stranded. Double-stranded compounds may also include protruding or non-complementary regions, wherein one or the other strand is single-stranded. Single-stranded compounds may include self-complementary regions, meaning that the compound can form a so-called "hairpin" or "stem-loop" structure with double-helix structural regions.

[0250] In some embodiments, the nucleic acid compound that inhibits the ActRII receptor may contain a nucleotide sequence complementary to a region consisting of the full-length ActRII receptor nucleic acid sequence or an activin nucleic acid sequence (e.g., activin A or activin B subunit, also known as β). A or β BThe nucleic acid sequence (the one containing the target transcript) may consist of no more than 1000, 500, 250, 100, or 50, 35, 30, 25, 22, 20, or 18 nucleotides. In one specific embodiment, the complementary region may be at least 8 nucleotides, optionally at least 10 or at least 15 nucleotides, optionally between 15 and 25 nucleotides. The complementary region may fall within an intron, the coding sequence of the target transcript, or a non-coding sequence, such as a coding portion. Typically, nucleic acid compounds that inhibit ActRII receptors may have a length of about 8 to about 500 nucleotides or base pairs, optionally about 14 to about 50 nucleotides. Nucleic acid compounds that inhibit ActRII receptors may be DNA (particularly used as antisense), RNA, or RNA:DNA hybrids. Either strand may include mixtures of DNA and RNA, as well as modified forms that are not readily classified as DNA or RNA. Similarly, double-stranded nucleic acid compounds can be DNA:DNA, DNA:RNA, or RNA:RNA, and either strand can also include a mixture of DNA and RNA, as well as modified forms that are not easily classified as DNA or RNA.

[0251] Nucleic acid compounds that inhibit ActRII receptors may include any of a variety of modifications, including one or more modifications to the backbone (the sugar-phosphate portion of the natural nucleic acid, including intermolecular bonds) or the base portion (the purine or pyrimidine portion of the natural nucleic acid). In some embodiments, the antisense nucleic acid compound may have a length of about 15 to about 30 nucleotides and will typically contain one or more modifications to improve certain properties, such as stability in serum, stability in cells, and stability at the site of possible delivery of the compound, for example, the stomach for oral delivery and the lungs for inhaled compounds. For RNAi constructs, the strand complementary to the target transcript is generally RNA or a modified form thereof. Other strands may be RNA, DNA, or any other variant. In some embodiments, the double helix portion of a double-stranded or single-stranded "hairpin" RNAi construct may have a length of 18 to 40 nucleotides, optionally about 21 to 23 nucleotides, as long as it functions as a Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymes or DNases and may also contain modified forms. In some implementations, under physiological conditions and at concentrations with little or no effect in nonsense or meaningful controls, nucleic acid compounds that inhibit the ActRII receptor can inhibit the expression of their target by approximately 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher, with concentrations testing the effect of the nucleic acid compounds including 1, 5, 10 micromoles or higher.

[0252] In other embodiments, the ActRII signaling inhibitors used in the compositions and methods described herein are antibodies. These antibodies include those that bind activins (particularly activin A or B subunits, also known as β-activins). A or β B Antibodies that bind to the ActRII receptor and disrupt its binding; and antibodies that bind to ActRII receptor peptides (e.g., soluble ActRIIA or soluble ActRIIB peptides) and disrupt activin binding.

[0253] Antisera or monoclonal antibodies against proteins / peptides can be generated using standard protocols with an immunogen derived from an ActRII receptor peptide or activin peptide (see, for example, *Antibodies: A Laboratory Manual* ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). Mammals, such as mice, hamsters, or rabbits, can be immunized with the immunogenic form of the ActRII receptor peptide, an antigenic fragment capable of evoking an antibody response, or a fusion protein. Techniques for conferring immunogenicity to proteins or peptides include conjugation with a vector or other techniques well known in the art. The immunogenic portion of the ActRII receptor or activin peptide can be administered in the presence of an adjuvant. Progression of immunization can be monitored by detecting antibody titers in plasma or serum. Antibody levels can be evaluated using the immunogen as an antigen and standard ELISA or other immunoassays.

[0254] After immunizing animals with an antigenic preparation of the ActRII receptor peptide, antiserum can be obtained, from which polyclonal antibodies can be isolated if necessary. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from the immunized animal and fused with immortalized cells (such as myeloma cells) using a standard somatic cell fusion procedure to produce hybridoma cells. Such techniques are well known in the art and include, for example, hybridoma techniques for producing human monoclonal antibodies (first developed by Kohler and Milstein, (1975) Nature, 256:495-497), human B-cell hybridoma techniques (Kozbar et al., (1983) Immunology Today, 4:72), and EBV hybridoma techniques (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Immunochemical screening of hybridoma cells can be used to produce antibodies that specifically react with ActRII receptor peptides, and monoclonal antibodies can be isolated from cultures containing such hybridoma cells.

[0255] The term "antibody" as used herein is intended to include fragments of an antibody that also specifically react with a target polypeptide. Antibodies can be fragmented using conventional techniques, targeting fragments for practical screening in the same manner described above for whole antibodies. For example, the F(ab)2 fragment can be generated by treating the antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bonds, producing the Fab fragment. Antibodies are intended to further comprise bispecific, single-chain, chimeric, humanized, and fully human molecules having an affinity for the ActRII receptor or activin polypeptide conferred by at least one CDR region of the antibody. Antibodies may further contain a detectable label linked thereto (e.g., the label may be a radioisotope, fluorescent compound, enzyme, or enzyme cofactor).

[0256] In some embodiments, the antibody is a recombinant antibody. This term encompasses any antibody partially generated through molecular biology techniques, including CDR-grafted or chimeric antibodies, human or other antibodies assembled from antibody domains selected from a library, single-chain antibodies, and single-domain antibodies (e.g., human V). H Protein or camel V HH (Protein). In some embodiments, the antibody may be a monoclonal antibody. For example, a method for generating a monoclonal antibody that specifically binds to an ActRII receptor polypeptide or an activin polypeptide may include administering a mouse a quantity of an immunogenic composition containing an antigenic polypeptide that effectively stimulates a detectable immune response; obtaining antibody-producing cells (e.g., cells derived from the spleen) from the mouse; fusing the antibody-producing cells with myeloma cells to obtain an antibody-producing hybridoma; and testing the antibody-producing hybridoma to identify a hybridoma that produces a monoclonal antibody that specifically binds to the antigen. Once the hybridoma is obtained, it can be proliferated in a cell culture, optionally under culture conditions in which the hybridoma-derived cells produce a monoclonal antibody that specifically binds to the antigen. The monoclonal antibody may be purified from the cell culture.

[0257] As is generally understood in the art, the adjective "reacts specifically with" when referring to antibodies means that the antibody has sufficient selectivity between the target antigen (e.g., the ActRII receptor peptide) and other non-target antigens, making the antibody useful at least in detecting the presence of the target antigen in a specific type of biological sample. In some methods employing antibodies, such as therapeutic applications, a high degree of binding specificity is desirable. Monoclonal antibodies generally tend to be more effective (compared to polyclonal antibodies) in distinguishing the desired antigen from the cross-reacting peptide. A characteristic affecting the specificity of antibody-antigen interactions is the antibody's affinity for the antigen. While desired specificity can be achieved at a variety of different affinities, generally preferred antibodies have an affinity of approximately 10. -6 10 -7 10 -8 10-9 Or even lower affinity (dissociation constant). Given the very tight binding between activin and the ActRII receptor, it is expected that neutralizing antiactivin or anti-ActRII receptor antibodies should generally have an affinity of 10. -10 Or a lower dissociation constant.

[0258] Furthermore, the technique used to screen antibodies for identification of desired antibodies can affect the properties of the resulting antibodies. For example, if the antibody is used to bind to an antigen in solution, then testing solution binding may be desirable. A variety of different techniques are available for testing the interaction between antibodies and antigens to identify particularly desired antibodies. These techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore™ binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., paramagnetic bead systems, IGEN International Inc., Gaithersburg, Md.), Western blotting, immunoprecipitation assays, and immunohistochemistry.

[0259] In some embodiments, the ActRII signaling inhibitors used in the compositions and methods described herein comprise alternative forms of activin, particularly those having altered type I receptor-binding domains that can bind type II receptors and cannot form an active ternary complex. In some embodiments, nucleic acids that inhibit activin A, B, C, or E, or particularly ActRII receptor expression, such as antisense molecules, siRNAs, or ribozymes, may be used in the compositions and methods described herein. In some embodiments, the ActRII signaling inhibitors used in the compositions and methods described herein exhibit selectivity for inhibiting GDF11-mediated signal transduction relative to other members of the TNF-β family, particularly for GDF8 and activin.

[0260] In other embodiments, the ActRII signaling inhibitors used in the compositions and methods described herein are non-antibody proteins with ActRII receptor antagonist activity, including statins (i.e., statin α subunits), follicle-stimulating hormone (FSH) (e.g., FSH-288 and FSH-315), Cerberus, FSH-related protein (“FSRP”), endoglin, activin C, α(2)-macroglobulin, and activin A with an M108A (methionine to alanine change at position 108) mutation.

[0261] In one specific embodiment, the ActRII signal transduction inhibitor used in the compositions and methods described herein is a follicle-inhibitory polypeptide that antagonizes activin bioactivity and / or binds to activin. The term "follicle-inhibitory polypeptide" includes such polypeptides as any naturally occurring polypeptide containing follicle-inhibitory and any variants (including mutant, fragment, fusion, and peptide mimic forms) that retain useful activity, further including any functional monomer or polymer of follicle-inhibitory. Variants of follicle-inhibitory polypeptides that retain activin-binding properties can be identified based on previous studies relating to the interaction between follicle-inhibitory and activin. For example, WO2008 / 030367 discloses a specific follicle-inhibitory domain (“FSD”) that exhibits importance for activin binding, and is incorporated herein by reference in its entirety. Follicle-stimulating hormone (FSH) peptides include peptides derived from any known FSH peptide sequence, having at least about 80% identity with the sequence of a FSH peptide, optionally with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher identity. Examples of FSH peptides include shorter isotypes or other variants of mature FSH peptides or human FSH precursor peptides, such as those described in WO2005 / 025601, which are incorporated herein by reference in their entirety.

[0262] In one specific embodiment, the ActRII signaling inhibitor used in the compositions and methods described herein is a follicle-stimulating hormone-inhibitor-like gene (FLRG) that antagonizes activin biological activity and / or binds to activin. The term "FLRG polypeptide" includes any naturally occurring polypeptide containing FLRG and any variants (including mutants, fragments, fusions, and peptide mimics) that retain useful activity. FLRG polypeptide variants that retain activin-binding properties can be identified using conventional methods for determining FLRG and activin interactions. See, for example, U.S. Patent No. 6,537,966, which is incorporated herein by reference in its entirety. FLRG polypeptides include polypeptides derived from any known FLRG sequence, having at least about 80% sequence identity with a FLRG polypeptide sequence, optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher.

[0263] In some embodiments, functional variants or modified forms of the follicle-stimulating hormone (FSH) peptide and the FLRG peptide comprise fusion proteins having at least a portion of the FSH peptide or the FLRG peptide and one or more fusion domains, said fusion domains being, for example, domains that promote the separation, detection, or stabilization or polymerization of the peptide. Suitable fusion domains have been discussed in detail above with reference to the ActRII and ActRIIB peptides. In one embodiment, the ActRII signaling inhibitor is a fusion protein comprising an activin-binding moiety of the FSH peptide fused to an Fc domain. In another embodiment, the ActRII signaling inhibitor is a fusion protein comprising an activin-binding moiety of the FLRG peptide fused to an Fc domain.

[0264] 7.7 Determination Method The ability of various ActRII peptide variants or soluble ActRII peptide variants to inhibit ActRII can be tested. Furthermore, the ability of compounds to inhibit ActRII can be tested. Once an inhibitor of ActRII signaling activity is confirmed, these compounds can be used in the methods described herein. ActRII can be ActRIIA or ActRIIB. The following assays are described for ActRIIA, but similar methods can be used for ActRIIB.

[0265] 7.7.1 Reference Population In some embodiments, the size of the reference population can be 1, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, or 1000 individuals. In some embodiments, the reference population consists of randomized volunteers. In some embodiments, the reference population consists of healthy individuals. In some embodiments, the reference population consists of individuals of the same age, weight, and / or sex as the patient population described in Section 7.5. In some embodiments, the reference population consists of individuals who do not have β-thalassemia.

[0266] 7.7.2 Evaluate protein levels and / or activity The levels of proteins such as hemoglobin, fetal hemoglobin, or GDF11 can be determined by any method known in the art or described herein. For example, the levels of proteins such as hemoglobin, fetal hemoglobin, or GDF11 in a tissue sample can be determined by evaluating (e.g., quantifying) the transcribed RNA of the protein in the sample, using techniques such as RNA blotting, PCR analysis, real-time PCR analysis, or any technique known in the art or described herein. In one embodiment, the levels of proteins in a tissue sample can be determined by evaluating (e.g., quantifying) the mRNA of the protein in the sample.

[0267] The levels of proteins such as hemoglobin, fetal hemoglobin, or GDF11 in tissue samples can also be determined by evaluating (e.g., quantifying) the protein expression levels of proteins in the sample, using techniques such as immunohistochemical analysis, Western blotting, ELISA, immunoprecipitation, flow cytometry, or any technique known in the art or described herein. In a specific embodiment, the protein level is determined by methods capable of quantifying the amount of protein present in a patient tissue sample (e.g., human serum) and / or capable of detecting the correction of protein levels after treatment with an activin type II receptor signal transduction inhibitor. In one embodiment, the protein level in a tissue sample is determined by evaluating (e.g., quantifying) the protein expression of proteins in the sample using an ELISA.

[0268] 7.7.3 Decreased serum ferritin levels Serum ferritin levels can be determined using methods known to those skilled in the art. Typically, adult males have serum ferritin concentrations between 24 and 336 ng / mL. Typically, adult females have concentrations between 11 and 307 ng / mL.

[0269] 7.7.4 Iron Level Iron levels, such as liver or myocardial iron levels, can be determined using methods known to those skilled in the art. For example, iron levels (e.g., liver iron concentration or myocardial iron concentration) can be determined by magnetic resonance imaging.

[0270] 7.7.5 Erythrocyte Morphology Red blood cell morphology can be evaluated using methods known to those skilled in the art (e.g., blood smears). The ratio of abnormal red blood cells to the total number of red blood cells in the subject can be determined as follows: for example, by obtaining a blood sample, performing a blood smear, calculating the number of abnormal red blood cells in the smear, calculating the total number of red blood cells in the smear, and obtaining the ratio by dividing the number of abnormal red blood cells in the smear by the total number of red blood cells. The ratio of basophilic stippled red blood cells to the total number of red blood cells in the subject can be determined as follows: for example, by obtaining a blood sample, performing a blood smear, calculating the number of red blood cells with basophilic stippling in the smear, calculating the total number of red blood cells in the smear, and obtaining the ratio by dividing the number of red blood cells with basophilic stippling in the smear by the total number of red blood cells. The ratio of atypical red blood cells to the total number of red blood cells in the subject can be determined as follows: for example, by obtaining a blood sample, performing a blood smear, calculating the number of atypical red blood cells in the smear, calculating the total number of red blood cells in the smear, and obtaining the ratio by dividing the number of atypical red blood cells in the smear by the total number of red blood cells. The ratio of cleavage cells to the total number of red blood cells in an object can be determined as follows: For example, obtain a blood sample, prepare a blood smear, count the number of cleavage cells in the smear, calculate the total number of red blood cells in the smear, and then calculate the ratio by dividing the number of cleavage cells in the smear by the total number of red blood cells. The ratio of irregularly contracted red blood cells to the total number of red blood cells in an object can also be determined as follows: For example, obtain a blood sample, prepare a blood smear, count the number of irregularly contracted red blood cells in the smear, calculate the total number of red blood cells in the smear, and then calculate the ratio by dividing the number of irregularly contracted red blood cells in the smear by the total number of red blood cells.

[0271] 7.7.6 Erythrocyte Reaction The duration of the red blood cell response in the subjects who achieved the response can be calculated. The algorithm used to calculate the duration of the response is as follows: (1) First response day = the first day of the first 12-week interval in which the response was displayed. Last response day = the last day of the last consecutive 129-week interval in which the response was displayed. Last evaluation date = the last follow-up date for subjects who continued treatment or the termination date for subjects who terminated treatment. The duration of the red blood cell response can be calculated as follows, depending on whether the response terminated before the last evaluation date: (1) For subjects whose response did not continue until the end of the treatment period and whose response duration was not deleted, the response duration is calculated as follows: Response duration = Last response day - First response day + 1; (2) For subjects who continued to display a red blood cell response at the end of the treatment period and whose response termination date was deleted, the response duration is calculated as follows: Response duration = Last response evaluation date - First response day + 1.

[0272] The time to the first red blood cell response can be calculated as follows: From the date of the first dose of study drug to the first day of the response, the following calculation can be used: Time to response = First response day - First study drug day + 1.

[0273] 7.7.7 Blood Transfusion Burden It is estimated that one unit of red blood cells contains approximately 200 mg of iron, while the body typically loses only 1.5 mg of iron per day. The transfusion burden of subjects treated according to the methods described herein can be determined by measuring their transfusion requirements (i.e., the amount and frequency of red blood cell transfusions). As a non-limiting example, if a subject requiring two units of red blood cells every three weeks reduces the transfusion frequency to every four weeks when treated according to the methods described herein, the subject's transfusion burden is reduced by 25%.

[0274] 7.7.8 Evaluation of clinical complications Extramedullary hematopoietic (EMH) masses in a subject can be evaluated using assays known to those skilled in the art, such as magnetic resonance imaging (MRI) and computed tomography (CT). In some embodiments, extramedullary hematopoietic masses in a subject can be evaluated using MRI.

[0275] Splenomegaly can be evaluated using methods known to those skilled in the art, such as magnetic resonance imaging (MRI).

[0276] Tricuspid regurgitation velocity (TRV) can be evaluated using methods known to those skilled in the art, such as echocardiography (ECHO).

[0277] The liver iron concentration of a subject can be evaluated using methods known to those skilled in the art, such as magnetic resonance imaging (MRI).

[0278] 7.7.9 Osteoporosis and bone mineral density Non-limiting examples of osteoporosis symptoms include back pain, height loss over time, kyphosis, easy fractures, and decreased bone mineral density. Bone mineral density in subjects treated according to the methods provided herein can be determined by methods known to those skilled in the art, such as bone mineral density scanning (also known as dual-energy X-ray absorptiometry (DXA or DEXA) or bone optical density measurement) and ultrasound. In some embodiments, bone mineral density in subjects treated according to the methods provided herein is determined by DXA.

[0279] 7.7.10 Skeletal deformities Skeletal deformities in subjects treated according to the methods provided herein can be determined by methods known to those skilled in the art, such as X-rays and imaging techniques, such as magnetic resonance imaging (MRI) and computed tomography.

[0280] 7.7.11 Bone Turnover Various circulatory markers of bone turnover can be used to diagnose bone disorders, such as low bone turnover. Circulatory markers of bone turnover include bone-specific alkaline phosphatase (bAP), osteocalcin, procollagen type I C-terminal propeptide (PICP), and insulin-like growth factor-1 (IGF-1); some are markers of bone resorption, such as pyridinoline, deoxypyridinoline, tartrate-resistant acid phosphatase (TRAP), type 5b TRAP, pyridinoline, deoxypyridinoline, and procollagen type I C-terminal telopeptide (ICTP), serum or urinary collagen crosslinks (N-terminal or C-terminal peptide), and 25-hydroxyvitamin D. Measurements of the entire parathyroid hormone (PTH) molecule can also be used. Skilled technicians are familiar with imaging methods that allow for the evaluation of bone mineral density (BMD), bone volume, trabecular bone volume, and trabecular bone thickness. See, for example, Tilman B. Drueke and Sharon M. Moe, Disturbances of bone and mineral metabolism in chronic kidney disease: an international initiative to improve diagnosis and treatment, Nephrol DialTransplant (2004) 19: 534-536; Okuno S, Inaba M., Biochemical markers of bone turnover. New aspect. Dialysis and bone metabolism marker, Clin Calcium. 2009Aug;19(8):1084-91; Herberth J, Monier-Faugere MC, Mawad HW, Branscum AJ,Herberth Z, Wang G, Cantor T, Malluche HH, The five most commonly used intact parathyroid hormone assays are useful for screening but not for diagnosing bone turnover abnormalities in CKD-5 subjects, Clin Nephrol. 2009 Jul;72(1):5-14; Lehmann G, Ott U, Kaemmerer D, Schuetze J, Wolf G., Bone histomorphometry and biochemical markers of bone turnover in subjects with chronic kidney disease stages 3 - 5, Clin Nephrol. 2008 Oct;70(4):296-305;Drüeke TB., Is parathyroid hormone measurement useful for the diagnosis ofrenal bone disease?, Kidney Int. 2008 Mar;73(6):674-6;Yamada S, Inaba M,Kurajoh M, Shidara K, Imanishi Y, Ishimura E, Nishizawa Y. Utility of serumtartrate-resistant acid phosphatase (TRACP5b) as a bone resorption marker in subjects with chronic kidney disease: A review from renal dysfunction., ClinEndocrinol (Oxf). 2008 Aug;69(2):189-96. Epub 2008 Jan 23. See also, Paul D.Miller, Diagnosis and Treatment of Osteoporosis in Chronic Renal Disease,2009。.

[0281] Another biomarker for monitoring bone resorption in CKD subjects with mild renal impairment is the serum concentration of type I collagen N-terminal peptide (S-NTX). See, for example, Hamano T, Fujii N, Nagasawa Y, Isaka Y, Moriyama T, Okada N, Imai E, Horio M, Ito T., Serum NTX is a practical marker for assessing antiresorptive therapy for glucocorticoid treated subjects with chronic kidney disease., Bone. 2006 Nov;39(5):1067-72. Epub 2006 Jun 16.

[0282] Quantitative computed tomography (QCT) can also be used to measure bone turnover.

[0283] Biomarkers such as Runx2 and Alp can be evaluated to monitor osteoblast transformation in subjects. Biomarkers such as Sm22-α can be evaluated to monitor vascular smooth muscle function and the level of differentiated vascular smooth muscle cells.

[0284] 7.7.12 Heart size and cardiomegaly Heart size and cardiac hypertrophy can be determined by any method known to a skilled technician, such as magnetic resonance imaging, electrocardiography, echocardiography, and non-contrast-enhanced cardiac computed tomography.

[0285] 7.7.13 Quality of Life To evaluate the quality of life of subjects treated according to the methods provided herein, the (36) Health Survey Short Form (SF-26) and / or the Functional Assessment of Cancer Therapy-Anemia (FACT-An) can be used.

[0286] The SF-36 (version 2.0) is a self-implemented tool consisting of eight multiple-item scales assessing the following eight health domains: (1) Physical Functioning (PF), 10 items from 3a to 3j; (2) Role-Body (RP), 4 items from 4a to 4d; (3) Body Pain (BP), items 7 and 8; (4) General Health (GH), items 1 and 11a to 11d; (5) Vitality (VT), items 9a, 9e, 9g, and 9i; (6) Social Functioning (SF), items 6 and 10; (7) Role-Emotion (RE), items 5a, 5b, and 5c; and (8) Mental Health (MH), 5 items 9b, 9c, 9d, 9f, and 9h. Two overall total scores are also available: (1) Physical Component Total Score (PCS); and (2) Mental Component Total Score (MCS). The health domain scores, along with the PCS and MCS scores, are converted into canonical scores (mean 50, SD 10), where higher scores indicate better health. The main benefit of the SF-36 is the canonical health domain scores and the canonical PCS and MCS scores. It allows for the evaluation of canonical health domain scores, PCS and MCS scores, and summary statistics (n, mean, standard deviation, median, minimum, and maximum) of these canonical scores from baseline. The scoring and handling of missing values ​​for the SF-36 can be completed by following the usage instructions provided by the tool developer.

[0287] Alternatively, FACT-An can be used to assess the quality of life of individuals treated according to the methods described herein. FACT-An is a 47-item cancer-specific questionnaire consisting of a core 27-item general questionnaire (FACT-General or FACT-G Total) measuring four general domains of quality of life (physical, social / family, emotional, and functional health). The FACT-An scale is formatted by subscale domain on pages 1-4 and is self-administered using a 5-point Likert scale (0 = none; 1 = a little; 2 = some; 3 = quite a lot; and 4 = very a lot). The total scale level of the FACT tool can be completed by following the instructions provided by the tool developer. The FACT-G Total score can be evaluated by summing the four domains of the general HRQoL tool.

[0288] 7.7.14 Common Naming Standards for Adverse Events (CTCAE, Version 4.0) Grade 1 refers to a minor adverse event. Specifically, Grade 1 refers to transient or mild discomfort. No activity restriction or medical intervention / therapy is required for Grade 1 adverse events. Grade 2 refers to a moderate adverse event. Specifically, Grade 2 refers to mild to moderate restriction of activity. Some support may be required; however, no or minimal medical intervention / therapy is required for Grade 2 adverse events. Grade 3 refers to a serious adverse event. Specifically, Grade 3 refers to significant restriction of activity. Some support and medical intervention / therapy are required, and hospitalization is possible for Grade 3 adverse events. Grade 4 refers to a life-threatening adverse event. Specifically, Grade 4 refers to extreme restriction of activity, requiring extensive support, extensive medical intervention / therapy, and hospitalization or a work hospital is possible for Grade 4 adverse events. Grade 5 is death.

[0289] 7.7.15 Hematocrit Hematocrit measures the percentage of red blood cells in a given volume of whole blood and can be included as part of a standard whole blood count. Normal hematocrit is approximately 45% for men and approximately 40% for women. However, patients with β-thalassemia often have a lower hematocrit than normal. Therefore, measuring hematocrit in β-thalassemia patients being treated according to the methods described herein allows for the assessment of the efficacy of such treatment.

[0290] 7.7.16 Hemoglobin Hemoglobin concentration can be determined according to methods known to those skilled in the art. Patients with β-thalassemia typically have hemoglobin concentrations lower than those commonly observed. Therefore, the determination of hemoglobin concentration in β-thalassemia patients being treated according to the methods provided herein allows for the assessment of the efficacy of such treatment.

[0291] 7.7.17 Screening assay The ability of various ActRII peptide variants or soluble ActRII peptide variants to inhibit ActRII can be tested. Furthermore, the ability of compounds to inhibit ActRII can be tested. Once an inhibitor of ActRII signaling activity is confirmed, these compounds can be used in the methods described herein. ActRII can be ActRIIA or ActRIIB. The following assays are described for ActRIIA, but similar methods can be used for ActRIIB.

[0292] For example, the effects of ActRIIA peptide variants on the expression of genes involved in bone formation or bone destruction can be evaluated. This can be performed, if desired, in the presence of one or more recombinant ActRIIA ligand proteins (e.g., activin), by transfecting cells to produce ActRIIA peptides and / or variants thereof, optionally producing ActRIIA ligands. Similarly, ActRIIA peptides can be administered to mice or other animals, and one or more bone properties, such as density or volume, can be evaluated. Fracture healing rates can also be evaluated. Dual-energy X-ray absorptiometry (DEXA) is a well-established, non-invasive, quantitative technique for evaluating bone mineral density in animals. In humans, central DEXA systems can be used to evaluate bone mineral density in the spine and pelvis. These are the best predictors of total bone mineral density. Peripheral DEXA systems can be used to evaluate bone mineral density in peripheral bones, including, for example, the bones of the hand, wrist, ankle, and foot. Conventional X-ray imaging systems, including CAT scans, can be used to evaluate bone growth and fracture healing. Furthermore, bone mineral density can be measured using quantitative computed tomography (qCT). The mechanical strength of bone can also be evaluated.

[0293] In some respects, this article provides the use of ActRIIA peptides (e.g., soluble ActRIIA peptides) and activin peptides to identify compounds (acting agents) that function as agonists or antagonists of the activin-ActRIIA signaling pathway. Compounds identified through this screening can be tested to evaluate their ability to regulate bone growth or mineralization in vitro. Optionally, these compounds can be further tested in animal models to evaluate their ability to regulate tissue growth in vivo.

[0294] There are various methods for screening therapeutic agents that regulate tissue growth by targeting activin and ActRIIA peptides. In some embodiments, high-throughput screening of compounds can be performed to identify agents that interfere with the activin or ActRIIA-mediated effects on bone. In some embodiments, assays are performed to screen or identify compounds that specifically inhibit or reduce the binding of ActRIIA peptides to activin. Alternatively, assays can be used to identify compounds that enhance the binding of ActRIIA peptides to activin. In further embodiments, compounds can be identified by their ability to interact with activin or ActRIIA peptides.

[0295] Multiple assay methods will be sufficient, and those not explicitly described herein will be readily apparent to those skilled in the art based on this disclosure. As described herein, the test compounds (actuators) used herein can be prepared by any combinatorial chemical method. Alternatively, the subject compound can be a naturally occurring biomolecule synthesized in vivo or in vitro. For example, compounds (actuators) to be tested for their ability as tissue growth regulators can be produced, for example, by bacteria, yeast, plants, or other organisms (e.g., natural products), chemically produced (e.g., small molecules, including peptide mimics), or recombinantly produced. The test compounds contemplated herein include non-peptide organic molecules, peptides, polypeptides, peptide mimics, sugars, hormones, and nucleic acid molecules. In one specific embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2000 Daltons.

[0296] Test compounds may be provided as single discrete entities or as larger, more complex libraries, for example, prepared through combinatorial chemistry. These libraries may contain, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other organic compound classes. Test compounds provided to the test system may be in isolated forms or as mixtures of compounds, particularly in the initial screening steps. Optional compounds may be derivatized from other compounds and have derivatizing groups that facilitate compound isolation. Non-limiting examples of derivatizing groups include biotin, luciferin, digitalisin, green fluorescent protein, isotopes, multiple histidines, magnetic beads, glutathione S-transferase (GST), photoactivated crosslinking agents, or any combination thereof.

[0297] In many drug screening procedures involving libraries of tested compounds and natural extracts, high-throughput assays are desirable to maximize the number of compounds investigated within a specified timeframe. Assays performed, for example, in cell-free systems where purified or semi-purified proteins are available, are often preferred for “preliminary” screening because they allow for rapid development and relatively easy detection of alterations in molecular targets mediated by the test compound. Furthermore, the effects of cytotoxicity and / or bioavailability of the test compound are generally negligible in in vitro systems, and the assay focuses primarily on the drug’s effect on the molecular target, as may be evident in alterations in binding affinity between ActRIIA peptides and activins.

[0298] For illustrative purposes only, in an exemplary screening assay, the target compound is contacted with isolated and purified ActRIIA peptides, which typically bind to activin. A composition containing ActRIIA ligands is then added to a mixture of the compound and the ActRIIA peptide. Detection and quantification of the ActRIIA / activin complex provides a means of determining the ability of a compound to inhibit (or enhance) the formation of a complex between the ActRIIA peptide and activin. The efficacy of the compound can be assessed by constructing dose-response curves from data obtained using test compounds at various concentrations. Furthermore, control assays can be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified activin is added to a composition containing the ActRIIA peptide, and the formation of the ActRIIA / activin complex is measured in the absence of the test compound. It should be understood that the order in which the reactants are mixed is generally variable and can be mixed simultaneously. Additionally, cell extracts and lysates can be used instead of purified proteins to provide a suitable cell-free assay system.

[0299] The formation of the complex between the ActRIIA peptide and the activin can be detected using a variety of techniques. For example, the regulation of complex formation can be quantitatively determined by immunoassay or chromatography using detectable labeled proteins, such as radiolabeled (e.g., 32P, 35S, 14C, or 3H), fluorescently labeled (e.g., FITC), or enzyme-labeled ActRIIA peptides or activins.

[0300] In some embodiments, fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays are contemplated herein when directly or indirectly measuring the extent of the interaction between the ActRIIA peptide and its binding protein. Furthermore, other detection methods, such as those based on optical waveguides (PCT Publication WO 96 / 26432 and U.S. Patent No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many of the embodiments described herein.

[0301] Furthermore, interaction trap assays, also known as "two-hybrid assays," can be used to identify agents that disrupt or enhance the interaction between ActRIIA peptides and their binding proteins. See, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696. In one specific embodiment, it is contemplated herein that a reverse two-hybrid system is used to identify compounds (e.g., small molecules or peptides) that disrupt the interaction between ActRIIA peptides and their binding proteins. See, for example, Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and U.S. Patent Nos. 5,525,490, 5,955,280 and 5,965,368.

[0302] In some embodiments, the subject compound is identified by its ability to interact with ActRIIA or the activin peptide. The interaction between the compound and the ActRIIA or activin peptide can be covalent or non-covalent. For example, at the protein level, such interactions can be identified using in vitro biochemical methods, including photocrosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46:1). In some cases, compounds can be screened using mechanism-based assays, such as assays that detect compounds binding to activin or ActRIIA peptides. This can include solid-phase or liquid-phase binding events. Alternatively, the gene encoding the activin or ActRIIA peptide can be transfected into cells along with a reporter gene system (e.g., β-galactosidase, luciferase, or green fluorescent protein), and the library can be screened, preferably by high-throughput screening or by screening individual members of the library. Other mechanism-based binding assays can be used, such as assays that detect changes in free energy. Binding assays can be performed using targets immobilized in pores, beads, or chips, captured by immobilized antibodies, or separated by capillary electrophoresis. The bound compounds are typically detected using colorimetry, fluorescence, or surface plasmon resonance.

[0303] In some respects, this article provides methods and agents for regulating (stimulating or inhibiting) bone formation and improving bone quality. Therefore, any compound identified can be tested in vitro or in vivo in intact cells or tissues to confirm its ability to regulate bone growth or mineralization. Various methods known in the art can be utilized for this purpose. In particular, the ability of a compound to improve bone turnover can be tested.

[0304] For example, the effect of ActRIIA or activin peptides or test compounds on bone or cartilage growth can be determined by measuring Msx2 induction or differentiation of osteoprogenitor cells into osteoblasts in a cell-based assay (see, for example, Daluiski et al., Nat Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Other examples of cell-based assays include analyzing the osteogenic activity of the subject ActRIIA or activin peptides and test compounds in mesenchymal progenitor cells and osteoblasts. For example, recombinant adenoviruses expressing activin or ActRIIA peptides can be constructed to infect pluripotent mesenchymal progenitor C3H10T1 / 2 cells, pre-osteoblastic C2Cl2 cells, and osteoblastic TE-85 cells. Osteogenic activity was then determined by measuring alkaline phosphatase induction, osteocalcin, and matrix mineralization (see, for example, Cheng et al., J bone Joint Surg Am. 2003, 85-A(8):1544-52).

[0305] This article also provides in vivo assays for measuring bone or cartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86 (2001) disclosed a rat model of osteoporosis in which early bone repair after fracture was studied. Kubo et al., Steroid Biochemistry & Molecular Biology, 68:197-202 (1999) also disclosed a rat model of osteoporosis in which late bone repair after fracture was studied. Andersson et al., J. Endocrinol. 170:529-537 described a mouse model of osteoporosis in which ovariectomy caused significant loss of bone mineral content and mineral density, with a loss of approximately 50% of bone mineral density in trabecular bone. Bone density in ovariectomized mice could be increased by administering factors such as parathyroid hormone. In some respects, fracture healing assays known in the art can be used. These assays include fracture techniques, histological analysis, and biomechanical analysis, described, for example, in U.S. Patent No. 6,521,750, the entire contents of which are incorporated herein by reference to the disclosure of their experimental protocols relating to the causes and measurement of the extent and repair process of fractures.

[0306] 7.8 Combination Therapy In some embodiments, the methods described herein are combined with a second pharmaceutically active agent. Such combination therapies can be achieved by administering the therapeutic components simultaneously, sequentially, or individually. Furthermore, when administered as a component of such a combination therapy, the ActRII signaling inhibitor and the second pharmaceutically active agent can be synergistic, thereby reducing the daily dose of either or both components compared to the dose of either component typically administered as a monotherapy. Alternatively, when administered as a component of such a combination therapy, the ActRII signaling inhibitor and the second pharmaceutically active agent described herein can be additive, thereby ensuring that the daily dose of each component is similar or the same compared to the dose of either component typically administered as a monotherapy.

[0307] In some embodiments, the ActRII signaling inhibitor provided herein is administered on the same day as a second active pharmaceutical agent or therapy. In some embodiments, the ActRII signaling inhibitor is administered one, two, three, or more days before the second active pharmaceutical agent or therapy. In some embodiments, the ActRII signaling inhibitor is administered one, two, three, or more days after the second active pharmaceutical agent or therapy. In some embodiments, the ActRII signaling inhibitor is administered within one, two, three, or more weeks after the second active pharmaceutical agent or therapy.

[0308] In some embodiments, the second pharmaceutical active agent or therapy is an active agent or therapy for treating β-thalassemia. Non-limiting examples of pharmaceutical active agents or therapies for treating β-thalassemia include red blood cell transfusion, iron chelation therapies such as deferoxamine, deferoxone and / or deferasirox, fetal hemoglobin inducers such as hydroxyurea, and hematopoietic stem cell transplantation.

[0309] 7.9 Pharmaceutical Compositions In some embodiments, an ActRII signaling inhibitor (e.g., an ActRII peptide) is formulated together with a pharmaceutically acceptable carrier for the methods described herein. For example, the ActRII peptide may be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compound may be formulated for administration in any convenient manner for human or veterinary use. ActRII may be ActRIIA or ActRIIB.

[0310] In a preferred embodiment, the ActRII signal transduction inhibitor is formulated for subcutaneous administration.

[0311] In another preferred embodiment, the ActRII signaling inhibitor is packaged in a container as a sterile, preservative-free lyophilized powder or block. In some embodiments, the container contains 25 mg of the ActRII signaling inhibitor. In some embodiments, the container containing 25 mg of the ActRII signaling inhibitor contains a total of 37.5 mg of protein. In some embodiments, the ActRII signaling inhibitor in the container containing 25 mg of the ActRII signaling inhibitor is reconstituted with 0.68 mL of water for injection. In some embodiments, the container contains 75 mg of the ActRII signaling inhibitor. In some embodiments, the container containing 75 mg of the ActRII signaling inhibitor contains a total of 87.5 mg of protein. In some embodiments, the ActRII signaling inhibitor in the container containing 75 mg of the ActRII signaling inhibitor is reconstituted with 1.6 mL of water for injection. In some embodiments, the ActRII signaling inhibitor in the container is reconstituted with a volume of water for injection such that the final concentration of the reconstituted ActRII signaling inhibitor in the water for injection is 50 mg / mL, with a pH of approximately 6.5. In some embodiments, the ActRII signaling inhibitor is administered to the subject within 10 hours of reconstitution. In some embodiments, the container contains a 50 mg / mL concentration of ActRII signaling inhibitor in a 10 mM citrate-based buffer solution, wherein the 10 mM citrate-based buffer solution contains 10 mM citrate (pH 6.5), 9% sucrose, and 0.02% polysorbate 80. In some embodiments, the container is stored between 2°C and 8°C. In some embodiments, the container is stored between 2°C and 8°C for 18 months. In some embodiments, the container is a 3 mL glass vial with a gray butyl-coated stopper. In some embodiments, the container is a 3 mL glass vial with a gray rubber stopper. In some embodiments, the rubber stopper is secured in place by a corrugated aluminum flip-top with a colored plastic button. In some embodiments, the 3 mL glass vial contains 25 mg of the ActRII signal transduction inhibitor, and the colored plastic button is red. In some embodiments, the 3 mL glass vial contains 75 mg of the ActRII signal transduction inhibitor, and the colored plastic button is white.

[0312] In one specific embodiment, the ActRII signal transduction inhibitor is packaged in a container as a sterile, preservative-free lyophilized powder or block. In one specific embodiment, the container contains 50 mg / mL of the ActRII signal transduction inhibitor in 10 mM citrate buffer (pH 6.5). In one specific embodiment, the container contains 56 mg of the ActRII signal transduction inhibitor, 0.19 mg of citrate monohydrate, 3.03 mg of trisodium citrate dihydrate, 0.24 mg of polysorbate 80, and 100.80 mg of sucrose.

[0313] In some embodiments, the treatment methods provided herein include systemic or topical administration of the composition (containing an ActRII signaling inhibitor) as an implant or device. When administered, the treatment composition for the purposes provided herein is in a pyrogen-free, physiologically acceptable form. Therapeutic agents other than ActRII antagonists that may optionally be included in the above compositions may be administered concurrently or sequentially with the subject compound (e.g., ActRII peptides, such as ActRIIA and / or ActRIIB peptides (see Section 7.6)).

[0314] Typically, ActRII signaling antagonists are administered parenterally. In a preferred embodiment, ActRII signaling antagonists are administered subcutaneously. Pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRII peptides combined with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions, emulsions, or sterile powders, said sterile powders which may be reconstituted just before use into sterile injectable solutions or dispersions, which may contain antioxidants, buffers, antibacterial agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners. Examples of suitable aqueous and non-aqueous carriers that may be used in pharmaceutical compositions used in the methods described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, appropriate flowability can be maintained by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0315] The compositions described herein may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Protection against microbial activity can be achieved by including various antibacterial and antifungal agents, such as p-hydroxybenzoic acid, chlorobutanol, phenol, and sorbic acid. It may also be necessary to include isotonic agents, such as sugars and sodium chloride. Furthermore, prolonged absorption in injectable drug formulations can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0316] To understand the various factors that may affect the effects of the compounds described herein (e.g., ActRII peptides, such as ActRIIA and / or ActRIIB peptides (see Section 7.6), as described in Tables 1 and 2 of Section 7.3.2 above), the attending physician may determine the dosage regimen by considering the factors that may improve the effects of the compounds described herein (e.g., ActRII peptides, such as ActRIIA and / or ActRIIB peptides (see Section 7.6), as described in Tables 1 and 2 of Section 7.3.2 above).

[0317] In some embodiments, the ActRII signaling inhibitor in the pharmaceutical composition is substantially pure. Specifically, up to 20%, 10%, 5%, 2.5%, 1%, 0.1%, or up to 0.05% of the compound in the pharmaceutical composition is a compound other than the ActRII signaling inhibitor and a pharmaceutically acceptable carrier.

[0318] In some implementations, the ActRII signaling inhibitor is administered to the patient at room temperature, as described herein (e.g., as listed in Section 7.5).

[0319] 8. Examples 8.1 Example 1: A phase 3, double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of mactriib-fc in adults with transfusion-dependent β-thalassemia. This embodiment provides an overview of a phase 3, double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of ActRIIB-hFc (SEQ ID NO:25) in adults requiring regular red blood cell transfusions due to β-thalassemia. The indication for the phase 3 study was adults with transfusion-dependent β-thalassemia, diagnosed β-thalassemia or hemoglobin E / β-thalassemia, and those excluded from hemoglobin S / β-thalassemia.

[0320] 8.1.1 Objectives The primary objective of the Phase 3 study was to determine the proportion of subjects with a red blood cell response, defined as a reduction of ≥33% in transfusion burden (units of red blood cells over time) over 12 consecutive weeks after at least 6 months of treatment compared to placebo plus BSC at the 12-week interval prior to randomization with ActRIIB-hFc (SEQ ID NO:25) best supportive care (BSC).

[0321] Secondary objectives of the Phase 3 study include: (1) evaluating the safety and immunogenicity of ActRIIB-hFc (SEQ ID NO:25) relative to placebo; (2) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) on the proportion of subjects who have not received transfusions for ≥8 weeks relative to placebo; (3) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) on changes in liver iron concentration (LIC) relative to placebo; (4) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) treatment on quality of life (QoL) measures (e.g., the new transfusion-independent specific PRO, SF-36) relative to placebo; (5) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) on osteoporosis (bone mineral density) relative to placebo; and (6) evaluating ActRIIB-hFc (SEQ ID NO:25) on the proportion of subjects who have not received transfusions for ≥8 weeks relative to placebo. (7) To evaluate the mean percentage effect of ActRIIB-hFc (SEQ ID NO:25) on changes in transfusion burden relative to placebo plus BSC within the same 12-week period used in the primary endpoint analysis, compared to the 12-week interval before randomization; (8) To evaluate the duration of reduced transfusion burden or transfusion independence; (9) To evaluate the time to erythrocyte response; (10) To evaluate the effect of ActRIIB-hFc (SEQ ID NO:25) on changes in serum ferritin; (11) To evaluate the effect of ActRIIB-hFc (SEQ ID NO:25) on changes in cardiac iron overload; and (12) To evaluate the population pharmacokinetics (PK) of ActRIIB-hFc (SEQ ID NO:25) in subjects with β-thalassemia.

[0322] The exploratory objectives were: (1) to analyze the relationship between baseline and changes in serum GDF11 in patients who responded to treatment with ActRIIB-hFc (SEQ ID NO:25); and (2) to analyze the effect of ActRIIB-hFc (SEQ ID NO:25) on changes in fetal hemoglobin (HbF).

[0323] 8.1.2 Research Design This embodiment provides a phase 3, double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of ActRIIB-hFc (SEQ ID NO:25) (ACE-536) plus best supportive care (BSC) in adults with transfusion-dependent β-thalassemia. The study was divided into (i) a screening phase, (ii) a double-blind treatment phase, (iii) an open-label extension phase, and (iv) a follow-up phase.

[0324] Patient suitability was determined during a screening period that was 28 days prior to the first dose on the first day. Patients were classified based on the following factors: (1) baseline transfusion burden, of which high transfusion burden was ≥15 RBC units 24 weeks prior to randomization and low transfusion burden was 7–14 RBC units 24 weeks prior to randomization; and (2) geographic region.

[0325] During the treatment period, suitable subjects may be randomly assigned in a 2:1 ratio to either the experimental group (ActRIIB-hFc (SEQ ID NO:25)) plus BSC or the control group (placebo) plus BSC. The double-blind treatment period is considered to be the first 48 weeks following Day 1 of the study (i.e., the first dose on Day 1), independent of dose extension. Treatment with ActRIIB-hFc (SEQ ID NO:25) for each subject begins on Day 1 of the study. Subjects will begin treatment with approximately 0.8 mg / kg of ActRIIB-hFc (SEQ ID NO:25) administered subcutaneously (SC) every 3 weeks for 48 weeks. The dose of ActRIIB-hFc (SEQ ID NO:25) may be titrated up to a maximum of approximately 1.25 mg / kg.

[0326] During the treatment period and the extension period, the dose to the subject may be gradually increased from an initial dose of approximately 0.8 mg / kg ActRIIB-hFc (SEQ ID NO:25) to approximately 1 mg / kg ActRIIB-hFc (SEQ ID NO:25) and then to approximately 1.25 mg / kg ActRIIB-hFc (SEQ ID NO:25) until a dose change is required. In the first two cycles (i.e., the first 6 weeks), dose escalation may be based on transfusion frequency.

[0327] As described in Tables 1 and 2 above, and in accordance with the guidelines for dose variation, the dose of ActRIIB-hFc (SEQ ID NO:25) or placebo may be delayed and / or reduced for each subject.

[0328] All participants will be eligible to choose to be part of the open-label extension period and receive ActRIIB-hFc (SEQ ID NO:25) upon completion of the 48-week double-blind treatment period, at the investigator's discretion. The open-label extension period will last 96 weeks (i.e., 2 years) and will undergo dose escalation, dose variation, dose delay, and dose reduction as described in Tables 1 and 2 above. The extension period may be extended based on evolving safety data.

[0329] Subjects who complete the open-label extension period, are not recruited for the open-label extension period, or terminate treatment early will be transferred to the post-treatment follow-up period. The follow-up period will last for 12 weeks after the subject's last dose of study drug.

[0330] 8.1.2.1 Target Group The target population consisted of individuals diagnosed with transfusion-dependent β-thalassemia (including hemoglobin E / β-thalassemia), aged ≥18 years, and who were transfusion-dependent. Transfusion dependence was defined as receiving ≥7 red blood cell units every 24 weeks prior to randomization with a transfusion-free period of ≥35 days. In some respects, transfusion dependence was defined as receiving >6 red blood cell units every 24 weeks prior to randomization with a transfusion-free period of ≥35 days. In other respects, transfusion dependence was defined as receiving >5 red blood cell units every 24 weeks prior to randomization with a transfusion-free period of ≥35 days.

[0331] 8.1.2.2 Research Duration Participation in the study for each subject will last for approximately 160 weeks (40 months), including a screening period of up to 4 weeks (1 month), a placebo-controlled treatment period of 48 weeks (12 months), followed by an open-label extension period lasting approximately up to 96 weeks (2 years). Post-treatment follow-up will continue for 12 weeks (3 months) after the last dose.

[0332] Treatment completion for each individual subject is defined as the date of the last follow-up during the treatment period or the open-label extension period, whichever is the final date. Study completion is defined as the date of the last follow-up for each individual subject during the treatment period or the open-label extension period, whichever is the final date, plus 12 weeks after completion of the post-treatment follow-up period. Trial completion is defined as the date of the last follow-up after completion of treatment for the subject, or, as pre-specified in the protocol and / or statistical analysis plan, the date of receipt of the last data point for the last subject required for the primary, secondary, and / or exploratory analyses, whichever is the final date.

[0333] 8.1.2.3 Research on Treatment ActRIIB-hFc (SEQ ID NO:25) will be provided as a lyophilized powder, which will be reconstituted and administered to subjects via subcutaneous (SC) injection. If applicable, subcutaneous injection will be administered to the upper arm, abdomen, or thigh every 3 weeks during the treatment period and during the open-label extension period. Subjects will begin with ActRIIB-hFc (SEQ ID NO:25) at a dose level of approximately 0.8 mg / kg, and the dose may be escalated up to a maximum of approximately 1.25 mg / kg (see Tables 1 and 2 above).

[0334] Researchers can administer a placebo (saline solution) as a subcutaneous (SC) injection to subjects at a clinical site. Subcutaneous injections will be given every 3 weeks during the treatment period in the upper arm, abdomen, or thigh.

[0335] 8.1.2.4 Overview of Key Efficacy Evaluation The primary efficacy evaluation was the proportion of subjects who, at least 6 months after treatment, experienced a reduction in transfusion burden of ≥33% (per unit of red blood cells over time) in 12 consecutive weeks compared to placebo plus BSC with placebo plus BSC, as evaluated over a period of at least 6 consecutive weeks prior to randomization.

[0336] Secondary efficacy evaluations included: (1) the proportion of subjects who did not require transfusion for ≥ 8 weeks during treatment; (2) changes in liver iron concentration (LIC, mg / g dry weight) as measured by magnetic resonance imaging (MRI); (3) changes in quality of life (QoL; using TranQoL); and (4) changes in the mean daily dose of iron chelation therapy.

[0337] Other efficacy assessments may include: (1) total hip and lumbar spine bone mineral density as measured by DXA; (2) healthcare resource utilization; (3) change in percentage of transfusion burden using the same 12-week time as the primary endpoint; (4) duration of reduced transfusion burden or transfusion independence; (5) time to erythrocyte response; (6) changes in serum ferritin; and (7) changes in cardiac iron overload as measured by MRI; and changes in QoL as measured by SF-36. 8.1.2.5 Overview of Key Safety Assessments Safety can be evaluated in all patients by monitoring adverse events (AEs), clinical laboratory tests, vital signs, electrocardiograms (ECG), cardiac Doppler, anti-drug antibody (ADA) tests, and ECOG performance status.

[0338] 8.1.2.6 Overview of Key Exploratory Assessments The ability of ActRIIB-hFc (SEQ ID NO:25) to reduce serum GDF11 concentrations / levels and / or increase fetal hemoglobin concentrations / levels will be evaluated.

[0339] 8.2 Example 2: A phase 3 double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of ACTRIIB-HFC (SEQ ID NO:25) in adults with non-transfusion-dependent β-thalassemia. This embodiment provides an overview of a phase 3, double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of ACTRIIB-HFC (SEQ ID NO:25) in adults with non-transfusion-dependent β-thalassemia. The indication for the phase 3 study was adults with non-transfusion-dependent β-thalassemia diagnosed with β-thalassemia or hemoglobin E / β-thalassemia.

[0340] 8.2.1 Objectives The primary objective of the phase 3 study was to determine the role of ActRIIB-hFc (SEQ ID NO:25) in subjects diagnosed with transfusion-independent β-thalassemia, with a documented diagnosis of β-thalassemia or hemoglobin E / β-thalassemia, aged ≥18 years, who had received 0–6 units of red blood cells in the 24 weeks prior to randomization, and with a mean baseline hemoglobin level <10.0 g / dL. In some respects, subjects had received 0–5 units of red blood cells in the 24 weeks prior to randomization.

[0341] Secondary objectives of the Phase 3 study include: (1) evaluating the safety and immunogenicity of ActRIIB-hFc (SEQ ID NO:25) relative to placebo; (2) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) on changes in hepatic iron concentration (LIC) relative to placebo; (3) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) treatment on quality of life (QoL) measures (e.g., the novel transfusion-independent specific PRO, SF-36); and (4) evaluating the effect of ActRIIB-hFc (SEQ ID NO:25) treatment relative to placebo. (5) The effect of ActRIIB-hFc (SEQ ID NO:25) on the improvement of complications of thalassemia (when present), including extramedullary hematopoietic masses, leg ulcers, splenomegaly, pulmonary hypertension (PAH; measured by tricuspid regurgitation velocity (TRV)) and osteoporosis (measured by bone mineral density); (6) The effect of ActRIIB-hFc (SEQ ID NO:25) on changes in serum ferritin; (7) The effect of ActRIIB-hFc (SEQ ID NO:25) on the mean change in hemoglobin levels from baseline over consecutive 12-week intervals during treatment, relative to placebo; (8) The duration of erythrocyte response; and (9) The population pharmacokinetics (PK) of ActRIIB-hFc (SEQ ID NO:25) in subjects with β-thalassemia.

[0342] The exploratory objectives were: (1) to analyze the relationship between baseline and changes in serum GDF11 in patients who responded to treatment with ActRIIB-hFc (SEQ ID NO:25); (2) to analyze the effect of ActRIIB-hFc (SEQ ID NO:25) on changes in fetal hemoglobin (HbF); (3) to analyze the in vivo efficacy of ActRIIB-hFc (SEQ ID NO:25) on RBC quality; and (4) to analyze the effect of ActRIIB-hFc (SEQ ID NO:25) on the utilization of health resources.

[0343] 8.2.2 Research Design This embodiment provides a phase 3, double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of ACTRIIB-HFC (SEQ ID NO:25) (ACE-536) plus best supportive care in adults with non-transfusion-dependent β-thalassemia. The study was divided into (i) a screening phase, (ii) a double-blind treatment phase, (iii) an open-label extension phase, and (iv) a follow-up phase.

[0344] Patient suitability was determined during a screening period that took place 28 days prior to randomization. Patients were classified based on the following factors: (1) baseline hemoglobin level (≥ 8.5 g / dL or < 8.5 g / dL), and (2) ICT application.

[0345] During the treatment period, suitable subjects may be randomly assigned in a 2:1 ratio to either the experimental group (ActRIIB-hFc (SEQ ID NO:25)) plus BSC or the control group (placebo) plus BSC. The double-blind treatment period is considered to be the first 48 weeks following Day 1 of the study (i.e., the first dose on Day 1), independent of dose extension. Treatment with ActRIIB-hFc (SEQ ID NO:25) for each subject begins on Day 1 of the study. Subjects will begin treatment with approximately 0.8 mg / kg of ActRIIB-hFc (SEQ ID NO:25) administered subcutaneously (SC) every 3 weeks for 48 weeks. The dose of ActRIIB-hFc (SEQ ID NO:25) may be titrated up to a maximum of approximately 1.25 mg / kg.

[0346] During the treatment period and the extension period, the dose to the subject may be gradually increased from an initial dose of approximately 0.8 mg / kg ActRIIB-hFc (SEQ ID NO:25) to approximately 1 mg / kg ActRIIB-hFc (SEQ ID NO:25) and then to approximately 1.25 mg / kg ActRIIB-hFc (SEQ ID NO:25), unless dose changes are required. In the first two cycles (i.e., the first 6 weeks), dose escalation may be based on transfusion frequency.

[0347] As described in Tables 1 and 2 above, and in accordance with the guidelines for dose variation, the dose of ActRIIB-hFc (SEQ ID NO:25) or placebo may be delayed and / or reduced for each subject.

[0348] All participants will be eligible to choose to be part of the open-label extension period and receive ActRIIB-hFc (SEQ ID NO:25) upon completion of the 48-week double-blind treatment period, at the investigator's discretion. The open-label extension period will last 96 weeks (i.e., 2 years) and will undergo dose escalation, dose variation, dose delay, and dose reduction as described in Tables 1 and 2 above. The extension period may be extended based on evolving safety data.

[0349] Subjects who complete the open-label extension period, are not recruited for the open-label extension period, or terminate treatment early will be transferred to the post-treatment follow-up period. The follow-up period will last for 12 weeks after the subject's last dose of study drug.

[0350] 8.2.2.1 Target Group The target population consisted of individuals diagnosed with transfusion-independent β-thalassemia, with a documented diagnosis of β-thalassemia or hemoglobin E / β-thalassemia, aged ≥18 years, who had received 0–6 RBC units in the 24 weeks prior to randomization, and with a mean baseline hemoglobin level <10.0 g / dL. In some respects, participants had received 0–5 RBC units in the 24 weeks prior to randomization.

[0351] 8.2.2.2 Research Duration Participation in the study for each subject will last for approximately 160 weeks (40 months), including a screening period of up to 4 weeks (1 month), a placebo-controlled treatment period of 48 weeks (12 months), followed by an open-label extension period lasting approximately up to 96 weeks (2 years). Post-treatment follow-up will continue for 12 weeks (3 months) after the last dose.

[0352] Treatment completion for each individual subject is defined as the date of the last follow-up during the treatment period or the open-label extension period, whichever is the final date. Study completion is defined as the date of the last follow-up for each individual subject during the treatment period or the open-label extension period, whichever is the final date, plus 12 weeks after completion of the post-treatment follow-up period. Trial completion is defined as the date of the last follow-up after completion of treatment for the subject, or, as pre-specified in the protocol and / or statistical analysis plan, the date of receipt of the last data point for the last subject required for the primary, secondary, and / or exploratory analyses, whichever is the final date.

[0353] 8.2.2.3 Research on Treatment ActRIIB-hFc (SEQ ID NO:25) will be provided as a lyophilized powder, which will be reconstituted and administered to subjects via subcutaneous (SC) injection. If applicable, subcutaneous injection will be administered to the upper arm, abdomen, or thigh every 3 weeks during the treatment period and during the open-label extension period. Subjects will begin with ActRIIB-hFc (SEQ ID NO:25) at a dose level of approximately 0.8 mg / kg, and the dose may be escalated up to a maximum of approximately 1.25 mg / kg (see Tables 1 and 2 above).

[0354] Researchers can administer a placebo (saline solution) as a subcutaneous (SC) injection to subjects at a clinical site. Subcutaneous injections will be given every 3 weeks during the treatment period in the upper arm, abdomen, or thigh.

[0355] 8.2.2.4 Overview of Key Efficacy Evaluation The primary efficacy assessment is the proportion of subjects demonstrating the following red blood cell response: no transfusion required after a minimum 6-month treatment period relative to placebo plus BSC, and an increase in hemoglobin from a baseline of ≥1.0 g / dL (measured as the mean of hemoglobin values ​​over consecutive 12-week intervals). This assessment requires at least two hemoglobin measurements performed by a central laboratory at ≥1-week intervals over 4-week periods.

[0356] Secondary efficacy evaluations included: (1) changes in liver iron concentration (LIC, mg / g dry weight) as measured by magnetic resonance imaging (MRI); (2) changes in quality of life (QoL; new non-transfusion-dependent specific patient-reported outcomes (PRO)); (3) changes in daily dose of iron chelation therapy; (4) changes in serum ferritin concentration; (5) mean changes in hemoglobin from baseline over 12 weeks; (6) duration of mean hemoglobin increase from baseline ≥1.0 g / dL in the absence of transfusion; (7) pharmacokinetic parameters and exposure-response relationships; and (8) changes in one or more of the following pathological conditions: (i) extramedullary hematopoietic mass volume as measured by MRI; (ii) leg ulcer size; (iii) spleen volume as measured by MRI; (iv) TRV as measured by echocardiography; and (v) bone mineral density as measured by DXA.

[0357] 8.2.2.5 Overview of Key Safety Assessments Safety can be evaluated in all patients by monitoring adverse events (AEs), clinical laboratory tests, vital signs, electrocardiograms (ECG), cardiac Doppler, anti-drug antibody (ADA) tests, and ECOG performance status.

[0358] 8.2.2.6 Overview of Key Exploratory Assessments The efficacy of ActRIIB-hFc (SEQ ID NO:25) in reducing serum GDF11 concentrations / levels and / or increasing fetal hemoglobin concentrations / levels will be evaluated. Furthermore, the effects of ActRIIB-hFc (SEQ ID NO:25) treatment on erythrocyte quality will be evaluated. Finally, the effects of ActRIIB-hFc (SEQ ID NO:25) treatment on the utilization of health resources will be evaluated.

[0359] 8.3 Example 3: ActRiib-hfc (seq id no:25) signal transduction inhibitors increase hemoglobin, reduce transfusion burden, and decrease liver iron concentration in adults with β-thalassemia. 8.3.1 Introduction ActRIIB-hFc (SEQ ID NO:25), a fusion protein containing a modified activin receptor, is being developed for the treatment of β-thalassemia. In β-thalassemia, anemia and complications arise from excessive α-globin-driven ineffective erythropoiesis. ActRIIB-hFc (SEQ ID NO:25) binds to other ligands in the GDF11 and TGF-β superfamily to promote late erythrocyte differentiation. Non-clinical and clinical studies have shown that ActRIIB-hFc (SEQ ID NO:25) has a well-tolerated and corrective effect on ineffective erythropoiesis (Suragani R, Blood 2014, Attie K, Am J Hematol 2014).

[0360] This embodiment provides data from an ongoing phase 2 multicenter open-label dose-finding study to evaluate ActRIIB-hFc (SEQ ID NO:25) in adults with transfusion-dependent or non-transfusion-dependent β-thalassemia. Efficacy results included increased hemoglobin (Hb) in patients with non-transfusion-dependent β-thalassemia, reduced RBC infusion burden in patients with transfusion-dependent β-thalassemia, and liver iron concentration (LIC) obtained from magnetic resonance imaging (MRI).

[0361] 8.3.2 Method Inclusion criteria included individuals ≥ 18 years of age with transfusion-dependent or non-transfusion-dependent anemia and a baseline Hb < 10.0 g / dL. ActRIIB-hFc (SEQ ID NO: 25) was administered every 3 weeks up to 5 doses with a 2-month follow-up study. The time-series group (n=6 per group) was administered at doses of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.25 mg / kg. The extension group (n=30) is ongoing; patients who complete the study can be recruited into the ongoing 12-month extension study.

[0362] 8.3.3 Results Preliminary data (as of date) were available for 35 patients (25 non-transfusion-dependent and 10 transfusion-dependent) who received treatment for 3 months. The median patient age was 35.0 years (range 20–57 years), and 86% of patients had a history of splenectomy. The mean (SD) baseline Hb level for non-transfusion-dependent patients was 8.4 (±0.9) g / dL. The transfusion burden for transfusion-dependent patients ranged from 6–8 units / 12 weeks prior to treatment. Twenty patients were on stable iron chelation therapy (ICT) at baseline.

[0363] The mean (SD) increase in Hb in non-transfusion-dependent patients treated with 0.8–1.25 mg / kg ActRIIB-hFc (SEQ ID NO:25; n=8) was 1.7 g / dL, compared to 1.2 g / dL in patients treated with 0.2–0.6 mg / kg ActRIIB-hFc (SEQ ID NO:25; n=8). In the higher-dose group, 3 out of 6 patients (38%) showed a mean Hb increase >1.5 g / dL, lasting ≥2 weeks (mean duration 9 weeks), compared to zero in the lower-dose group. All 9 transfusion-dependent patients treated with 0.8–1.25 mg / kg ActRIIB-hFc (SEQ ID NO:25) experienced a >20% reduction in transfusion burden during the 12-week treatment period (mean 72%, range 43–100%) compared to pre-treatment levels.

[0364] In transfusion-dependent patients, despite iron chelation therapy, the mean baseline liver iron concentration was 7.4 mg Fe / g dry weight (n=9), and the mean reduction in liver iron concentration by week 16 of ActRIIB-hFc (SEQ ID NO:25) treatment was 16.3%. In non-transfusion-dependent patients with baseline liver iron concentration ≥5 mg / g dry weight, the mean reduction in liver iron concentration was 18.2% in patients receiving 0.6–1.25 mg / kg ActRIIB-hFc (SEQ ID NO:25; n=5), compared to 7.0% in patients receiving 0.2–0.4 mg / kg ActRIIB-hFc (SEQ ID NO:25; n=5). In non-transfusion-dependent patients with baseline liver iron concentration <5 mg / kg dry weight, the mean change in liver iron concentration was -1.2% (n=10). Three patients with long-standing leg ulcers at baseline (two non-transfusion dependent patients and one transfusion dependent patient) achieved substantial healing within 4–6 weeks after initiating ActRIIB-hFc (SEQ ID NO:25) treatment.

[0365] ActRIIB-hFc (SEQ ID NO:25) is generally well tolerated, and no serious adverse events have been reported to date. The most common associated adverse events include bone pain, headache, myalgia, limb pain, and weakness. No significant changes in platelets or white blood cells have been observed.

[0366] 8.3.4 Conclusion Subcutaneous administration of up to 5 doses of ActRIIB-hFc (SEQ ID NO:25) every 3 weeks is generally safe and well-tolerated, with elevated Hb levels in non-transfusion-dependent β-thalassemia patients and reduced transfusion requirements in transfusion-dependent β-thalassemia patients. Liver iron concentrations were significantly reduced in both transfusion-dependent and non-transfusion-dependent patients during treatment, and leg ulcer healing occurred in all 3 patients. ActRIIB-hFc (SEQ ID NO:25) is a promising therapy for patients with both transfusion-dependent and non-transfusion-dependent β-thalassemia.

[0367] 8.4 Example 4: ActRiib-hfc (seq id no:25) signal transduction inhibitors increase hemoglobin and reduce transfusion burden and decrease liver iron concentration in adults with β-thalassemia (continued) 8.4.1 Introduction See the Introduction (Section 8.3.1) and Materials and Methods (Section 8.3.2). This example provides additional data from Section 8.3, obtained at the last date of the Phase 2 study. Briefly, the dose escalation groups (35 patients in total) received 0.2–1.25 mg / kg (3–6 patients per group). Specifically, the dose escalation groups received doses of 0.2 (6 patients); 0.4 (6 patients); 0.6 (6 patients); 0.8 (6 patients); 1.0 (6 patients); and 1.25 mg / kg (5 patients). The extension group started at 0.8 mg / kg (4 patients; the dose level was increased to 1.0 mg / kg in 2 patients; possible doses up to 1.25 mg / kg). ActRIIB-hFc (SEQ ID NO:25) was administered subcutaneously every 3 weeks for up to 3 months. An extension study of an additional 12 months of treatment is ongoing. The primary efficacy endpoint is as follows. For non-transfusion-dependent patients (NTD; less than 4 U / 8 weeks, hemoglobin less than 10 g / dL): an increase in Hb of ≥ 1.5 g / dL for ≥ 2 weeks; for transfusion-dependent patients (TD; equal to or greater than 4 U / 8 weeks, confirmed for more than 6 months): a reduction in transfusion burden of ≥ 20% for more than 12 weeks. Secondary efficacy endpoints were liver iron concentration (measured by MRI), serum ferritin, and biomarkers of erythropoiesis.

[0368] 8.4.2 Results Preliminary data (as of date) were available for 39 patients (25 non-transfusion-dependent and 14 transfusion-dependent) who received ActRIIB-hFc (SEQ ID NO:25) for 3 months. Four patients continued treatment with ActRIIB-hFc (SEQ ID NO:25) for an extended 12-month period. The median age of the patients was 40.0 years (range 20–57 years), 49% were male, and 32% had a history of splenectomy. The mean (SD) baseline Hb of non-transfusion-dependent patients (NTD) was 8.3 (±0.9) g / dL. The mean liver iron concentration (by MRI) in NTD was 5.8 ±3.8 mg / g dw. Transfusion-dependent patients received a mean of 7.3 (±0.9) RBC units / 12 weeks and had a mean liver iron concentration (LIC) of 5.2 (±5.7) mg / g dw. As for LIC, the clinical goal is to maintain LIC below 5 mg / g dw in non-transfusion-dependent patients and 7 mg / g dw in transfusion-dependent patients.

[0369] Compared to zero patients out of 17 in the lower dose group (i.e., 0.2-0.6 mg / kg), in the higher dose group (i.e., 0.8-1.25 mg / kg), 4 out of 8 transfusion-independent patients (50%) showed a mean Hb increase >1.5 g / dL, which lasted for ≥2 weeks. Compared to zero patients out of 17 in the lower dose group (i.e., 0.2-0.6 mg / kg), in the higher dose group (i.e., 0.8-1.25 mg / kg), 3 out of 8 transfusion-independent patients (38%) showed a mean Hb increase >1.5 g / dL, which lasted for ≥9 weeks.

[0370] Of the 10 non-transfusion-dependent patients with a baseline LIC <5 mg / g day-w, 10 (100%) maintained a LIC <5 mg / g day-w. In 3 patients, the decrease in LIC during the 4-month treatment period ranged from approximately 0.5 mg / g day-w to approximately 2 mg / g day-w. In 2 patients, the increase in LIC during the 4-month treatment period ranged from approximately 0.5 mg / g day-w to approximately 1.0 mg / g, while in 5 patients, LIC remained essentially unchanged during the 4-month treatment period. Two patients receiving iron sequestrants showed a decrease in LIC of 0.5 mg / g day-w or less.

[0371] Of the 12 non-transfusion-dependent patients with a baseline LIC ≥ 5 mg / g day-w, 8 (67%) experienced a reduction of ≥ 1 mg / g day-w (between at least 1 mg / g day-w and up to 4.6 mg / g dry weight) during the 16-week treatment period. Five of these 8 patients received iron chelators during this period. Of the 8 patients, 5 experienced a reduction of approximately ≥ 2 mg / g day-w during the 16-week treatment period; 3 of these patients also received iron chelators. During the 16-week treatment period, 2 of the 12 patients experienced an increase of ≥ 1 mg / g day-w in LIC, and 1 patient experienced an increase of ≥ 2 mg / g day-w in LIC.

[0372] In non-transfusion-dependent patients, increased hemoglobin was found to be associated with decreased LIC (R 2 =0.305, p-value =0.063).

[0373] Of the 10 transfusion-dependent patients treated with ActRIIB-hFc (SEQ ID NO:25) at dose levels of 0.6–1.25 mg / kg over 12 weeks, 10 experienced a reduction in transfusion burden of >40%. Nine out of ten of these patients experienced a reduction of >60%, and two out of ten experienced a reduction of >80%.

[0374] Of the seven transfusion-dependent patients with a baseline LIC < 7 mg / g day-w, all seven (100%) maintained a LIC < 7 mg / g day-w during the 4-month ActRIIB-hFc (SEQ ID NO:25) treatment period. Five patients experienced a decrease in day-w from approximately 0.5 mg / g to approximately 2.0 mg / g, and two patients experienced an increase between approximately 0.5 mg / g and approximately 1.0 mg / g day-w during the 4-month ActRIIB-hFc (SEQ ID NO:25) treatment period. All seven patients also received iron chelators in addition to ActRIIB-hFc (SEQ ID NO:25).

[0375] Of the three transfusion-dependent patients with a baseline LIC ≥7 mg / g day-w, two experienced a reduction of ≥1 mg / g day-w (1.96 mg / g day-w and 4.7 mg / g day-w) during the 16-week ActRIIB-hFc (SEQ ID NO:25) treatment period. All three patients also received iron chelators in addition to ActRIIB-hFc (SEQ ID NO:25).

[0376] Of the three patients with chronic, persistent leg ulcers, all experienced healing when treated with ActRIIB-hFc (SEQ ID NO:25). One non-transfusion-dependent patient received ActRIIB-hFc (SEQ ID NO:25) at a dose of 0.4 mg / kg and experienced complete healing after 6 weeks. One transfusion-dependent patient received ActRIIB-hFc (SEQ ID NO:25) at a dose of 1.0 mg / kg and experienced complete healing after 18 weeks. One transfusion-dependent patient received ActRIIB-hFc (SEQ ID NO:25) at a dose of 1.25 mg / kg and experienced complete healing after 5 weeks.

[0377] ActRIIB-hFc (SEQ ID NO:25) is generally well tolerated, and no serious adverse events have been reported to date. The most common related adverse events include bone pain (23.1% of patients), myalgia (17.9% of patients), headache (15.4% of patients), weakness (10.3% of patients), limb pain (7.7% of patients), flu (5.1% of patients), spots (5.1% of patients), and musculoskeletal pain (5.1% of patients).

[0378] 8.4.3 Conclusion Subcutaneous administration of ActRIIB-hFc (SEQ ID NO:25) every 3 weeks for up to 16 weeks is generally safe and well-tolerated. In non-transfusion-dependent patients, >50% treated with higher doses of ActRIIB-hFc (SEQ ID NO:25; 0.8–1.25 mg / kg) showed a sustained increase in hemoglobin. A reduction in transfusion burden was observed in >33% of transfusion-dependent patients receiving ActRIIB-hFc (SEQ ID NO:25). Decreased liver iron concentrations were observed in most transfusion-dependent and non-transfusion-dependent patients with or without iron chelation therapy. Rapid healing of leg ulcers was observed in all three patients receiving ActRIIB-hFc (SEQ ID NO:25).

[0379] 8.5 Example 5: A phase 3, double-blind, randomized, placebo-controlled, multicenter study to determine the efficacy and safety of ACTRIIB-HFC (SEQ ID NO:25) relative to placebo in adults requiring regular red blood cell transfusions due to β-thalassemia. This example is an update to the overview (Section 8.1, 8.1) of the phase 3, double-blind, randomized, placebo-controlled, multicenter study (as described in Example 1) investigating the efficacy and safety of ACTRIIB-HFC (SEQ ID NO:25) in adults requiring regular red blood cell transfusions due to β-thalassemia. The indication for the phase 3 study was adults with transfusion-dependent β-thalassemia, a documented diagnosis of β-thalassemia or hemoglobin E / β-thalassemia, and who had excluded hemoglobin S / β-thalassemia.

[0380] 8.5.1 Brief Description This is a phase 3, double-blind, randomized, placebo-controlled, multicenter study that assesses the efficacy and safety of ACTRIIB-HFC (SEQ ID NO:25) plus best supportive care (BSC) relative to placebo plus BSC in adults who require regular red blood cell transfusions due to β-thalassemia.

[0381] The study was divided into a screening / adjustment period, a double-blind treatment period, a double-blind long-term treatment period, and a post-treatment follow-up period.

[0382] 8.5.2 Key Outcome Indicators The primary outcome measure of this study was the proportion of subjects who experienced hematological improvement (HI) from week 13 to week 14 compared to the 12 weeks prior to randomization. HI was defined as a decrease in red blood cell count (RBC) from baseline of ≥33% from week 13 to week 14 compared to week 12, with a reduction in transfusion burden of at least 2 units. Reports were made on the number of RBC units transfused from week 13 to week 14 and in the 12 weeks prior to randomization. This measurement was performed up to approximately week 24.

[0383] 8.5.3 Secondary Outcome Indicators The secondary outcome measure of this study was the proportion of subjects who experienced hematologic improvement (HI) from week 37 to week 48 compared to a 12-week interval prior to randomization. HI was defined as a ≥33% reduction in red blood cell (RBC) count from baseline from week 37 to week 48 compared to a 12-week interval, with a reduction in transfusion burden of at least 2 units, reported as the number of RBC units transfused from week 37 to week 48 and in the 12 weeks prior to randomization. The time range for this measure was up to approximately 48 weeks.

[0384] Another secondary outcome measure of this study was the proportion of participants who, for luspatercept plus BSC compared to placebo plus BSC, experienced a reduction of ≥50% in RBC count from baseline and a reduction in transfusion burden of at least 2 units from week 37 to week 48 compared to the 12-week interval before randomization. A reduction in transfusion burden of ≥50% was defined as a reduction of at least 2 units for luspatercept plus (best supportive care) BSC compared to placebo plus BSC from week 37 to week 48; reported as the number of RBC units transfused from week 37 to week 48 and in the 12 weeks prior to randomization. This measure spanned up to approximately 48 weeks.

[0385] Another secondary outcome measure of this study was the proportion of participants who, for luspatercept plus BSC versus placebo plus BSC, experienced a reduction of ≥50% in RBC count from baseline and a reduction of at least 2 units in transfusion burden from week 13 to week 14 compared to the 12-week interval before randomization, with the reduction of transfusion burden ≥50% defined ...

Claims

1. A method for treating β-thalassemia in a subject of need, the method comprising administering to the subject an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signal transduction inhibitor, wherein the activin receptor type II (ActRII) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days.

2. A method for treating transfusion-dependent β-thalassemia in a subject in need, the method comprising administering to the subject an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days.

3. A method for treating transfusion-independent β-thalassemia in a subject in need, the method comprising administering to the subject an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signal transduction inhibitor, wherein the activin receptor type II (ActRII) signal transduction inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days.

4. A method for treating β-thalassemia in a subject of need, the method comprising administering to the subject an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, wherein the subject's phenotype is selected from β-thalassemia. 0 / β 0 β + / β + β 0 / β + β 0 / HbE and β + / HbE.

5. A method for treating β-thalassemia in a subject of need, the method comprising administering an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously to the subject's upper arm, abdomen, or thigh every 21 days, wherein the subject's phenotype includes co-inherited two severe hemoglobin β-chain mutations, and wherein the subject suffers from α-thalassemia.

6. A method for treating β-thalassemia in a subject of need, the method comprising administering an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, wherein the activin receptor type II (ActRII) signaling inhibitor is administered subcutaneously in the subject's upper arm, abdomen, or thigh, wherein the subject's phenotype includes co-inherited two severe hemoglobin β-chain mutations, and wherein the subject suffers from hereditary fetal hemoglobin persistence.

7. A method for treating β-thalassemia in a subject of need, the method comprising administering to the subject an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor, followed by administering the subject of the ActRII signaling inhibitor once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh.

8. A method for treating β-thalassemia in a subject of need, the method comprising administering an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, followed by administering the ActRII signaling inhibitor to the subject once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the subject's phenotype is selected from β-thalassemia. 0 / β 0 β + / β + β 0 / β + β 0 / HbE and β + / HbE.

9. A method for treating β-thalassemia in a subject of need, the method comprising administering an initial dose of 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor to the subject, followed by administration of the ActRII signaling inhibitor once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the upper arm, abdomen, or thigh of the subject, and wherein the subject suffers from hereditary fetal hemoglobin persistence.

10. A method for treating β-thalassemia in a subject of need, the method comprising administering to the subject an initial dose of about 0.8 mg / kg or about 1.0 mg / kg of an activin receptor type II (ActRII) signaling inhibitor, followed by administering the subject of the ActRII signaling inhibitor once or more at 21-day intervals to treat β-thalassemia, wherein the administration comprises subcutaneous administration to the subject's upper arm, abdomen, or thigh, and wherein the administration is sufficient to detectably reduce the level of GDF-11 in the subject's serum between administrations.

Images