Activin-actrii antagonists and uses for treating anemia

[0001] Mature red blood cell, or erythrocyte, is responsible for carrying oxygen in vertebrate circulatory systems. Red blood cells carry high levels of hemoglobin, a protein that binds oxygen to the lungs at relatively high oxygen partial pressure (pO2) and supplies oxygen to areas of the body with relatively low pO2.

[0002] Mature red blood cells are produced from pluripotent hematopoietic stem cells in a process called erythropoiesis. In people in the postpartum period, erythropoiesis occurs mainly in the bone marrow and in the pulp of the red spleen. The coordinated action of different signaling pathways controls the balance of cell proliferation, differentiation, survival and death. Under normal conditions, red blood cells are produced at a rate that maintains a constant mass of red blood cells in the body, and production may increase or decrease in response to various stimuli, including increased or reduced oxygen pressure or tissue demand. The erythropoiesis process begins with the formation of a line of determined precursor cells and progresses through a number of distinct types of precursor cells. The final stages of erythropoiesis occur as reticulocytes are released into the bloodstream and lose their mitochondria and ribosomes, while adopting the morphology of mature red blood cells. Increased reticulocyte levels, or an increased reticulocyte: erythrocyte ratio in the blood indicates an increased rate of red blood cell production.

[0003] Erythropoietin (Epo) is widely recognized as the most important positive regulator of erythropoiesis in postpartum vertebrates. Epo regulates the compensatory erythropoietic response to reduced tissue oxygen pressure (hypoxia) and low levels of red blood cells or low levels of hemoglobin. In humans, elevated Epo levels stimulate red blood cell formation by stimulating the generation of erythroid progenitors in the bone marrow and spleen. In mice, Epo enhances erythropoiesis, mainly in the spleen.

[0004] Various forms of recombinant Epo are used by doctors to increase red blood cell levels in a number of clinical systems, and particularly to treat anemia. Anemia is a broadly defined condition characterized by lower than normal levels of hemoglobin or red blood cells. In some cases, anemia is caused by a primary disorder in the production or survival of red blood cells. More often, anemia is secondary to diseases of other systems (Weatherall & Provan (2000) Lancet 355, 1169-1175). Anemia may be the result of a reduced rate of production or an increased rate of destruction of red blood cells or loss of red blood cells due to bleeding. Anemia can arise from a number of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis and bone marrow transplantation.

[0005] Treatment with Epo typically results in an increase in hemoglobin concentration of about 13 g / dL in healthy people over a period of weeks. When administered to patients with anemia, this treatment regimen often provides a significant increase in hemoglobin and red blood cell levels and leads to improved quality of life and prolonged survival. Epo is not uniformly effective, and many individuals are resistant to even high doses (Horl et al. (2000) Nephrol Dial Transplant 1 5, 43-50). More than 50% of cancer patients have an inadequate response to Epo, approximately 10% with end-stage renal disease are hyporeactive (Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998) J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplastic syndrome respond positively (Estey (2003) Curr Opin Hematol 10, 60-67). Some factors, including inflammation, iron and vitamin deficiency, insufficient dialysis, aluminum toxicity, and hyperparathyroidism can predict a poor therapeutic response, the molecular mechanisms of Epo resistance are as yet unknown.

[0006] Thus, the purpose of the disclosure is to provide alternative compositions and methods for raising red blood cell levels in patients.

BRIEF DESCRIPTION OF THE INVENTION [0007] In a first aspect, the invention provides an activin-ActRII antagonist for use in treating or preventing anemia in a human patient in need thereof, wherein the activin-ActRII antagonist is a polypeptide having an amino acid sequence selected from the group in which consists of:

a) the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence at least 90% or 95% identical to SEQ ID NO: 2;

b) the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence at least 90% or 95% identical to SEQ ID NO: 3;

c) a polypeptide containing at least 50 consecutive amino acids selected from

SEQ ID NO: 2;

d) the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence at least 90% or 95% identical to SEQ ID NO: 16;

e) the amino acid sequence of SEQ ID NO: 17 or the amino acid sequence at least 90% or 95% identical to SEQ ID NO: 17;

f) a polypeptide comprising at least 50 consecutive amino acids selected from SEQ ID NO: 16;

g) the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence at least 95% identical to SEQ ID NO: 7;

h) the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence at least 95% identical to SEQ ID NO: 12;

i) the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence at least 95% identical to SEQ ID NO: 20; and

j) the amino acid sequence of SEQ ID NO: 21 or the amino acid sequence at least 95% identical to SEQ ID NO: 21.

[0008] The antagonist may be a fusion protein including, in addition to the activin-ActRII antagonist polypeptide, one or more parts of the polypeptide that enhance one or more of: in vivo stability, in vivo half-life, administration / administration, localization or distribution in tissue, protein complexing and / or purification. More specifically, the fusion protein may comprise a portion of the polypeptide selected from the group consisting of: immunoglobulin Fc domain and serum albumin.

[0009] An activin or ActRII antagonist polypeptide to be used according to the invention may comprise one or more modified amino acid residues, selected from: glycosylated amino acid, PEGylated amino acid, farnesylated amino acid, acetylated amino acid, biotinylated amino acid, lipid-linked amino acid and conjugated amino acid organic derivatizing agent.

[0010] Anemia is anemia that can be associated with multiple myeloma, a patient's chronic kidney disease, chemotherapeutic treatment of the patient, myelodysplastic syndrome or thalassemia.

[0011] In a second aspect, the invention provides a method of identifying an agent that increases red blood cell levels, the method comprising:

a) contacting the subject agent with an isolated ActRII polypeptide that is typically capable of binding to activin; b) adding a composition containing activin;

c) quantifying the efficacy of the agent in inhibiting the formation of a complex between the ActRII polypeptide and activin; and

d) assessing the effect of the agent on red blood cell levels in the animal.

[0012] In a third aspect, the invention provides an ActRII-Fc fusion protein for use in the treatment or prevention of anemia in a human patient in need thereof, wherein the ActRII-Fc fusion protein comprises an amino acid sequence selected from the group consisting of:

a) the amino acid sequence of SEQ ID NO: 3 or a sequence that is at least 90% or 95% identical to SEQ ID NO: 3,

b) the amino acid sequence of SEQ ID NO: 2 or a sequence that is at least 90% or 95% identical to SEQ ID NO: 2,

c) the amino acid sequence of SEQ ID NO: 7 or a sequence that is at least 90% or 95% identical to SEQ ID NO: 7,

d) the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence at least 95% identical to SEQ ID NO: 12,

e) the amino acid sequence of SEQ ID NO: 17 or a sequence that is at least 90% or 95% identical to SEQ ID NO: 17,

e) the amino acid sequence of SEQ ID NO: 16 or a sequence that is at least 90% or 95% identical to SEQ ID NO: 16,

f) the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence at least 90% or 95% identical to SEQ ID NO: 20, and

g) the amino acid sequence of SEQ ID NO: 21 or the amino acid sequence at least 95% identical to SEQ ID NO: 21.

[0013] Protein may cause less than 15% increase in patient's skeletal muscle mass. In one embodiment, the protein is administered to reach a patient's serum concentration of at least 100 ng / ml for a period of about 20 to 30 days. The protein may be administered to reach a patient's serum concentration in the range of 100 ng / ml to 1000 ng / ml. The protein may have a serum half life between 15 and 30 days. In some embodiments, the protein is to be administered to the patient no more than once a week, preferably no more than once a month.

[0014] In part, the disclosure demonstrates that activin antagonists as well as ActRIIa and ActRIIb antagonists can be used to increase red blood cell and hemoglobin levels. In particular, the disclosure demonstrates that the soluble form of ActRIIa acts as an inhibitor of activin and, when administered in vivo, increases red blood cell levels. A milder effect was seen with the soluble form of ActRIIb, which binds activin A with lower affinity than soluble ActRIIa. While soluble ActRIIa and ActRIIb can affect red blood cell levels through a mechanism other than antagonizing activin, the disclosure demonstrates that the desired therapeutic agents can be selected based on the activity of the activin antagonist or ActRIIa antagonist or both. Such agents are collectively referred to as activin-ActRII antagonists. Therefore, in some embodiments, the invention provides activin-ActRII antagonists, including, for example, activin-binding ActRIIa polypeptides, and activin-binding ActRIIb polypeptides, for use in increasing red blood cell levels and hemoglobin levels in patients and for treating related disorders with low levels of red blood cells or hemoglobin levels in patients who require it. As described in US Patent Application 2007-0249022A1, activinActRIIa antagonists can be used to stimulate bone growth and increase bone density. According to the description, the effects of such antagonists on red blood cell levels are faster and occur at lower doses than the effects of such antagonists on bone. Thus, in some embodiments, activin-ActRIIa antagonists can be used to increase levels of red blood cells or hemoglobin without causing a significant increase in bone density, for example, less than 3%, 5%, 10% or 15% increase in bone density. This selective effect can be achieved by using, for example, lower doses of activin-ActRIIa antagonist, less frequent dosing, or by using activin-ActRIIa antagonist with a shorter serum half-life, at dosages and frequencies calculated to provide lower serum concentrations.

[0015] Polypeptides useful in the invention may include a soluble, activin-binding ActRII polypeptide that binds to activin. The activin-binding polypeptide may be an ActRIIa polypeptide or an ActRIIb polypeptide. ActRII polypeptides can be formulated as a pharmaceutical preparation comprising an activin binding ActRII polypeptide and a pharmaceutically acceptable carrier. An activin binding ActRII polypeptide can bind to activin, with a KD of less than 1 micromolar or less than 100, 10 or 1 nanomolar. Optionally, the activin binding ActRII polypeptide selectively binds activin versus GDF11 and / or GDF8, optionally to KD, which is at least 10-fold, 20-fold or 50-fold lower relative to activin than relative to GDF11 and / or GDF8. Without wishing to be bound by a particular mechanism of action, this degree of selectivity for inhibition of activin over GDF11 / GDF8 inhibition is expected to be responsible for bone or erythropoiesis without consistently measurable muscle effects. In many embodiments, the ActRII polypeptide may be selected to cause less than 15%, less than 10%, or less than 5% of muscle growth at doses that achieve the desired effect on red blood cell levels. The composition may be at least 95% pure, relative to other polypeptide components, as estimated by size exclusion chromatography, and more preferably, the composition is at least 98% pure. The activin-binding ActRIIa polypeptide for use in the invention can be any of those disclosed above, such as a polypeptide with an amino acid sequence selected from SEQ ID NOs: 2, 3, 7 or 12, or with an amino acid sequence that is at least 90%, 95% , 97% or 99% identical to the amino acid sequence selected from SEQ ID NO: 2, 3, 7, 12 or 13. An activin binding ActRIIa polypeptide may comprise a functional fragment of a natural ActRIIa polypeptide, such as containing at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 1-3 or SEQ ID NO: 2, lacking 10 to 15 C-terminal amino acids ( "tail"). The activin-binding ActRIIb polypeptide for use in the invention can be any of those disclosed above, such as a polypeptide with an amino acid sequence selected from SEQ ID NOs: 16, 17, 20 or 21, or with an amino acid sequence that is at least 90%, 95% , 97% or 99% identical to the amino acid sequence selected from SEQ ID NO: 16, 17, 20 or 21. An activin binding ActRIIb polypeptide may comprise a functional fragment of a natural ActRIIb polypeptide, such as containing at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 15-12, or a sequence lacking 10 to 15 C-terminal amino acids ("tail"), such as SEQ ID NO: 17.

[0016] The soluble, activin-binding ActRII polypeptide may contain one or more changes in the amino acid sequence (e.g., in the ligand binding domain) relative to the naturally occurring ActRII polypeptide. Examples of altered ActRIIa and ActRIIb polypeptides are provided in WO 2006/012627, pp. 59-60 and pp. 55-58, respectively. A change in the amino acid sequence may, for example, alter the glycosylation of the polypeptide when it is produced in a mammalian, insect or other eukaryotic cell, or change the proteolytic cleavage of the polypeptide relative to the naturally occurring ActRII polypeptide. An activin-binding ActRII polypeptide useful in the invention can be a fusion protein that has, as one domain, an ActRII polypeptide (e.g. the ligand-binding portion of ActRIIa or ActRIIb) and one or more additional domains that provide the desired property, such as improved pharmacokinetics, easier purification, targeting to specific tissues, etc. For example, a fusion protein domain may enhance one or more of: in vivo stability , in vivo half-life, tissue uptake / administration, tissue localization or distribution, protein complex formation, multimerization of the fusion protein, and / or purification. The activin-binding ActRII fusion protein may contain an Fc (wild-type or mutant) immunoglobulin domain or serum albumin or other portion of the polypeptide that provides desirable properties such as improved pharmacokinetics, improved solubility or improved stability. In a preferred embodiment, the ActRIIa-Fc fusion comprises a relatively unstructured linker located between the Fc domain and the extracellular ActRII domain. This unstructured linker may correspond roughly to the 15-amino acid unstructured region at the C-terminus of the extracellular domain of ActRII ("tail"), or it may be an artificial sequence of 1, 2, 3, 4 or 5 amino acids or from 5 to 15, 20, 30, 50 or more amino acids that are relatively free of secondary structures, or a mixture of both. The linker may be rich in glycine and proline residues, and may, for example, contain a single threonine / serine and glycine sequence or repeating threonine / serine and glycine sequence (e.g., TG4 (SEQ ID NO: 22), or SG4 (SEQ ID NO: 23) singlets or repetitions). The fusion protein may contain a purification sub-sequence, such as an epitope tag, FLAG tag, polyhistidine sequence and GST fusion. Optionally, the soluble ActRII polypeptide includes one or more modified amino acid residues, selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, a lipid-linked amino acid and an organic derivatized conjugated amino acid. The pharmaceutical preparation may also contain one or more additional compounds, such as a compound that is used to treat bone disorders. Preferably, the pharmaceutical preparation is substantially pyrogen free. In general, it is preferred that the ActRII protein is expressed in a mammalian cell line that properly regulates the natural glycosylation of the ActRII protein, so as to reduce the likelihood of an adverse immune response in the patient. Human and CHO cell lines have been used successfully, and other popular mammalian expression systems are expected to be useful.

[0017] As used herein, ActRIIa proteins designated ActRIIa-Fc (a form with a minimal linker between the ActRIIa part and the Fc part) have the desired properties, including selective binding to activin vs. GDF8 and / or GDF11, ligand binding with high affinity and serum half-life of more than two weeks in animal models. The specification discloses pharmaceutical preparations containing ActRIIa-Fc polypeptides and a pharmaceutically acceptable excipient.

[0018] Also disclosed herein are nucleic acids encoding a soluble ActRII-activin-binding polypeptide, such as an ActRIIa or ActRIIb polypeptide. An isolated polynucleotide may comprise a sequence coding for a soluble activin-binding ActRII polypeptide as described above. For example, an isolated nucleic acid may contain a sequence coding for an extracellular domain (e.g. ligand binding domain) of ActRII and a sequence that will encode part or all of the transmembrane domain and / or cytoplasmic domain of ActRII, but outside the stop codon located within the transmembrane domain or cytoplasmic domain, or placed between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated polynucleotide may comprise a full-length ActRII polynucleotide sequence, such as SEQ ID NO: 4 or 5, or a full-length ActRIIb polynucleotide sequence, such as SEQ ID NO: 18, or a partially abbreviated version of ActRIIa or ActRIIb, an isolated polynucleotide additionally containing a transcript termination codon at least six hundred nucleotides before the 3 'end, or otherwise arranged such that translation of the polynucleotide will result in the formation of an extracellular domain, optionally conjugated to the full-length truncated portion of ActRII . The nucleic acid sequence for ActRIIa is SEQ ID NO: 14. The nucleic acids disclosed herein may be operably linked to an expression promoter, and the disclosure provides cells transformed with such recombinant polynucleotides. Preferably, the cell is a mammalian cell, such as a CHO cell.

[0019] Methods for producing a soluble, activin-binding ActRII polypeptide may include expressing any nucleic acid (e.g., SEQ ID NO: 4, 5, 14, 18 or 19) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cell . Such a method may include: a) culturing the cells under conditions suitable for expression of the soluble ActRII polypeptide in which the cell is transformed with the soluble ActRII expression construct; and b) recovering the soluble ActRII polypeptide thus expressed. Soluble ActRII polypeptides can be recovered as crude, partially purified or highly purified fractions. Purification can be accomplished by a series of purification steps, including, for example, one, two or three or more of the following, in any order: protein A chromatography, anion exchange chromatography (e.g. Q sepharose), hydrophobic interaction chromatography (e.g. phenylsepharose) , size exclusion chromatography and cation exchange chromatography.

[0020] The activin-ActRII antagonist disclosed herein, such as soluble activin-binding ActRIIa polypeptide, or soluble activin-binding ActRIIb polypeptide can be used in a method to stimulate red blood cell production or increase red blood cell levels in an individual. Disclosed are methods of treating a disorder associated with low red blood cell counts or low hemoglobin levels (e.g. anemia), or to stimulate red blood cell production in patients who require it. The method may comprise administering to a subject in need thereof an effective amount of an activin-ActRII antagonist. In some aspects, uses of activin-ActRII antagonists in the manufacture of a medicament for treating a disorder or condition as described herein are disclosed.

[0021] In some aspects, the disclosure provides a method for identifying an agent that stimulates the production of red blood cells. The method includes: a) identifying a test agent that binds to the activin or ligand binding domain of the ActRII polypeptide; and b) assessing the effect of the agent on levels of red blood cells, hemoglobin and / or levels of precursor red blood cells (e.g. reticulocyte levels).

BRIEF DESCRIPTION OF THE DRAWINGS [0022]

Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak, as visualized on a size separation column (left panel) and SDS-PAGE stained with Coomassie (right panel) (left path: molecular weight standards; right path: ActRIIa-hFc).

Figure 2 shows binding of ActRIIa-hFc to activin and GDF-11 as measured by the Biacore ™ assay.

Figure 3 shows the effect of ActRIIa-hFc on the number of red blood cells in non-human female primates. Female cynomolgus monkeys (four groups, five monkeys each) were treated with placebo or 1 mg / kg, 10 mg / kg or 30 mg / kg ActRIIa-hFc on day 0, day 7, day 14 and day 21. Figure 3A shows the number of red blood cells (RBC). Figure 3B shows hemoglobin levels. Statistical significance was shown relative to baseline for each treatment group. On day 57, two monkeys remained in each group.

Figure 4 shows the effect of ActRIIa-hFc on the number of red blood cells in non-human primates. Male cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1mg / kg, 10 mg / kg or 30 mg / kg ActRIIahFc on day 0, day 7, day 14 and day 21. Figure 4A shows the number of red blood cells (RBC ). Figure 4B shows hemoglobin levels. Statistical significance was shown relative to baseline for each treatment group. On day 57, two monkeys remained in each group.

Figure 5 shows the effect of ActRIIa-hFc on reticulocyte counts in non-human female primates. Cynomolgus monkeys (four groups, five monkeys each) were treated with placebo or 1 mg / kg, 10 mg / kg or 30 mg / kg ActRIIa-hFc on day 0, day 7, day 14 and day 21. Figure 5A shows the absolute number of reticulocytes . Figure 5B shows the percentage of reticulocytes relative to RBC. Statistical significance was shown relative to baseline for each group. On day 57, two monkeys remained in each group.

Figure 6 shows the effect of ActRIIa-hFc on reticulocyte counts in non-human female primates. Cynomolgus monkeys (four groups, five monkeys each) were treated with placebo or 1 mg / kg, 10 mg / kg or 30 mg / kg ActRIIa-hFc on day 0, day 7, day 14 and day 21. Figure 6A shows the absolute number of reticulocytes . Figure 6B shows the percentage of reticulocytes relative to RBC. Statistical significance was shown relative to baseline for each group. On day 57, two monkeys remained in each group.

Figure 7 shows the results from a human clinical study as described in Example 5, where the area under the curve (AUC) and the administered ActRIIa-hFc dose have a linear correlation, regardless of whether ActRIIa-hFc was administered intravenously (IV) or subcutaneously (SC) .

Figure 8 shows a comparison of serum ActRIIa-hFc levels in patients after IV or SC administration.

Figure 9 shows bone alkaline phosphatase (BAP) levels in response to different dose levels of ActRIIa-hFc. BAP is a marker for anabolic bone growth.

Figure 10 shows the median change from baseline of hematocrit levels from a human clinical study described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated doses.

Figure 11 shows the median change from baseline of hemoglobin levels from a human clinical study described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated doses.

Figure 12 shows the median change from baseline in RBC (red blood cell) counts from the human clinical study described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated doses.

Figure 13 shows the median change from baseline in reticulocyte counts from the human clinical study described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated doses.

DETAILED DESCRIPTION OF THE INVENTION

1. Summary [0023] The transforming growth factor beta (TGF-beta) superfamily contains a number of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on many different types of cells in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development, in creating tissue patterns and specifications, and may affect a number of differentiation processes, including fat deposition, muscle formation, cartilage formation, heart development, blood cell formation, nerve formation and cell differentiation. epithelial cells. The family is divided into two main branches: branches BMP / GDF and TGF-beta / Activin / BMP, whose members have different, often complementary effects. By manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in the body. For example, cattle breeds Piedmontese and Belgian Blue carry the mutation of the function loss type GDF8 (also called myostatin), which causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17 (1): 71-4. In addition, in humans, inactive GDF8 alleles are associated with increased muscle mass and, apparently, exceptional strength. Schuelke et al., N Engl J Med 2004, 350: 2682-8.

[0024] Activins are dimeric polypeptide growth factors that belong to the TGF-beta superfamily. There are three main forms of activin (A, B and AB) that are homo / heterodimers of two closely related β subunits (β, respectively_Αβ_Α, β_Ββ_Β and eAeB). The human genome also codes for activin C and activin E, which are mainly expressed in the liver, and heterodimeric forms are also known_C or e_E. In the TGF-beta superfamily, activins are unique and multi-functional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, affect cell cycle progression, positively or negatively, depending on the type of cell, and induce mesodermal differentiation, which least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198: 500-512; Dyson et al., 1997, Curr Biol. 7: 81-84; Woodruff, 1998, Biochem Pharmacol. 55: 953 -963). In addition, erythroid differentiation factor (EDF) isolated from stimulated human monocytic leukemia cells was found to be identical to activin A (Murata et al., 1988, PNAS, 85: 2434). Activin A has been suggested to stimulate bone marrow erythropoiesis. In several tissues, the associated heterodimer, inhibin, antagonist activin signaling. For example, during the release of follicle stimulating hormone (FSH) from the pituitary gland, activin stimulates FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that may regulate activin bioactivity and / or bind to activin include folistatin (FS), follistatin-related protein (FSRP), and₂-makroglobulinę.

[0025] TGF-β signaling is mediated by heteromeric type I and type II serine / threonine kinase receptor complexes that phosphorylate and activate the underlying Smad protein after ligand stimulation (Massague, 2000, Nat. Rev.

Moth. Cell Biol. 1: 169-178). These type I and type II receptors are transmembrane proteins composed of a ligand-binding extracellular domain, with a cysteine-rich region, transmembrane domain and cytoplasmic domain, with predicted serine / threonine specificity. Type I receptors are necessary for signaling; and type II receptors are required for ligand binding and for expression of type I receptors. Activin type I and II receptors form a stable complex upon ligand binding, resulting in phosphorylation of type I receptors by type II receptors.

[0026] Two related type II receptors (ActRII), ActRIIa and ActRIIb have been identified as type II receptors for activins (Mathews and Vale, 1991, Cell 65: 973-982; Attisanoet al.,

1992, Cell 68: 97-108). In addition to activins, ActRIIa and ActRIIb may interact biochemically with several other TGF-β family proteins, including BMP7, Nodal, GDF8 and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130: 217-226; Lee and 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 primary type I receptor for activins, especially for activin A, and ALK-7 may well serve as an activin receptor, especially for activin B.

[0027] As demonstrated herein, soluble ActRIIa polypeptide (sActRIIa), which shows a significant preference for binding to activin A, unlike other members of the TGF-beta family, such as GDF8 or GDF11, is effective in stimulating red blood cell levels in vivo. Without wishing to be associated with any particular mechanism, the sActRIIa effect is expected to be caused mainly by the activin antagonist effect, taking into account the very strong activin binding (picomolar dissociation constant) exhibited by the specific sActRIIa construct used in these studies. Regardless of the mechanism, it is evident from the disclosure that ActRIIa-activin antagonists increase red blood cell levels in rodents, monkeys and humans. It should be noted that hematopoiesis is a complex process, regulated by a number of factors, including erythropoietin, G-CSF and iron homeostasis. The terms "increase in red blood cells" and "stimulate red blood cell formation" refer to clinically measurable values, such as hematocrit, red blood cell counts, and hemoglobin, and are intended to be indifferent to the mechanism by which such changes occur.

[0028] As also demonstrated herein, soluble ActRIIb (sActRIIb) polypeptide is effective in increasing reticulocyte levels in vivo, which effect is expected to increase hematocrit in the long term.

[0029] Data cited herein for non-human primates are reproducible in mice, rats as well as humans and therefore, ActRII polypeptides and other activin-ActRII antagonists can be used to stimulate red blood cell production and increase in red blood cell levels in mammals , from rodents to humans. Activin-ActRII antagonists include, for example, activin-binding soluble ActRIIa polypeptides, activin-binding soluble ActRIIb polypeptides, antibodies that bind to activin (particularly activin A or B subunits, also referred to as eA or eB) and interfere with ActRIIa and / or binding ActRIIb, antibodies that bind to ActRIIa and interfere with activin binding, antibodies that bind to ActRIIb and interfere with activin binding. non-antibody proteins selected for binding to activin, ActRIIa or ActRIIb (see e.g. WO / 2002/088171, WO / 2006/055689 and WO / 2002/032925, for examples of such proteins and methods of designing and selecting the same), randomized peptides selected for binding ActRIIa or ActRIIb activin, often attached to the Fc domain. Two different proteins (or other moieties) with activin binding activity, ActRIIa or ActRIIb, especially activin binding substances blocking type I binding sites (e.g. soluble type I activin receptor) and type II (e.g. soluble type II activin receptor), respectively, can be linked together to form a bifunctional binding molecule. Nucleic acid aptamers, small molecules and other agents that inhibit the activin-ActRIIa signaling axis are included as activin-ActRII antagonists. Different proteins have activin-ActRII antagonist activity, including inhibin (i.e. inhibin alpha subunit), although inhibin does not antagonize activin universally, in all tissues, folistatin (e.g. folistatin-288 and folistatin315), FSRP, activin C, afa (2) - macroglobulin and M108A (change of methionine to alanine at position 108) ) activin A. mutant In general, alternative forms of activin, especially those with changes in the binding domain of the type I receptor may bind to type II receptors and are unable to form an active tertiary complex, thus acting as antagonists. In addition, nucleic acids such as antisense molecules, siRNAs or ribozymes that inhibit activin A, B, C or E, or, particularly, expression of ActRIIa or ActRIIb can be used as activin-ActRII antagonists. The activin-ActRIIa antagonist to be used may show specificity in inhibiting activin-mediated signaling, vs. other members of the TGF-beta family, especially relative to GDF8 and GDF11.

[0030] The terms used in this specification generally have their usual meanings in a given field, within the scope of the invention, and in the specific context in which each term is used. Some concepts are discussed below or elsewhere in the specification to provide additional guidance to the practitioner by describing the compositions and methods of the invention and their preparation and use. The scope or meaning of any use of a term will be apparent from the particular context in which the term was used.

[0031] "Approximately" and "approximately" are generally intended to mean an acceptable level of error in a measured quantity, taking into account the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.

[0032] Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values ​​that are within the order of magnitude, preferably within 5 times, and more preferably within 2 times a given value. The numerical quantities given in the description are approximations, unless otherwise stated, which means that the term "approximately" or "approximately" can be deduced when not explicitly marked.

[0033] Sequences can be compared with each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically include polymer sequence overlays, e.g., using sequence overlay programs and / or algorithms that are well known in the art (e.g., BLAST, FASTA and MEGALIGN, to name a few). One of skill in the art can readily understand that, in such overlays, where the mutation contains an insertion or deletion of a residue, the superimposition of the sequence will introduce a "gap" (typically indicated by a dash or "A") in the polymer sequence containing no introduced or missing residue.

[0034] "Homologous" in all its grammatical forms and spelling variants, refers to the relationship between two proteins that have "common evolutionary origin", including superfamily proteins in the same species of organism, as well as homologous proteins from different species of organisms. Such proteins (and the nucleic acids encoding them) have sequence homology, which is reflected in their sequence similarity, whether in terms of percent identity, or the presence of specific residues or motifs and conservative positions.

[0035] The term "sequence similarity", in all its grammatical forms, refers to the degree of identity or agreement between nucleic acid or amino acid sequences that may or may not have a common evolutionary origin.

[0036] However, in common use and the same application, the term "homologous" when modified with an adverb such as "highly" may refer to sequence similarity and may or may not refer to common evolutionary origin.

2. ActRII polypeptides [0037] In some aspects, the invention relates to certain ActRII polypeptides for use in treating or preventing anemia. As used herein, the term "ActRII" refers to the type II activin receptor family. This family includes both type IIa activin receptor and type IIb activin receptor.

[0038] As used herein, the term "ActRIIa" refers to proteins of the type IIa activin receptor family (ActRIIa) from any species and variants derived from such ActRIIa proteins by mutagenesis or other modification. A reference to ActRIIa in the description is understood to be a reference to any of the currently identified forms. Family members

ActRIIa are generally transmembrane proteins, consisting of a ligand-binding extracellular domain, with a cysteine-rich region, transmembrane domain and cytoplasmic domain, with predicted serine / threonine kinase activity.

[0039] The term "ActRIIa polypeptide" includes polypeptides comprising any naturally occurring polypeptide - member of the ActRIIa family, as well as any variants thereof (including mutants, fragments, fusions and peptidomimetic forms) that retain useful activity. See, for example, WO / 2006/012627. For example, ActRIIa polypeptides include polypeptides derived from the sequence of any known ActRIIa having a sequence at least about 80% identical to the sequence of the ActRIIa polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity. For example, the ActRIIa polypeptide of the invention may bind to and inhibit the function of the ActRIIa protein and / or activin. The ActRIIa polypeptide can be selected for activity in stimulating red blood cell formation in vivo. Examples of ActRIIa polypeptides include the human ActRIIa polypeptide precursor (SEQ ID NO: 1) and soluble human ActRIIa polypeptides (e.g., SEQ ID NOs: 2, 3, 7 and 12).

[0040] The sequence of the human ActRIIa precursor protein is as follows:

MGΑΑΛΚ LA FA V FLI SC SSG AILGRS ETQECLFFNANWEKDRTgQTGVE P CTGDKDKRRRCFATWKgISGSΙΐIVKCGCWLDDINCYDRTDCVEKKDSP EVYFCC CEGNMCNEKFSY FPEMEVTQPT SNPVTP KPPYY Ν PLΝIL

KLIAGIVICAFWVYRHNKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLE VKARGRFGCUMKAQLLNEYVAVKIFPIQDKQSWQNEY EV ¥ SLPGMKHEN ILQ FIGAEKRGT S VDVDLW LIT A FH EKGSLSDFLK AN WS WNELCHIAE T MAR GLAYLH E DI P GLK DG HK PAIΞ HR DIKSKNVL1KNN LTAC and AD FGL AL K FiE AGKSAG DT HGQVGTRRYMń PE 7LEGAIN fQR DA FL RIDMYAMG L VLMELASRCTAA DGPVDEYML P FEEEIGQH PSLE DWOEWVHKKKR PVL RDYHQK HAGM AMLC ETIEECWDHDAEARLSAGCWGERITQMQRLT Ν11T TEDIVTVVTMVTNVDFPPKESSL (SEQ ID NO: 1) [0041] The signal peptide was underlined once; the extracellular domain is in bold, and the potential N-linked glycosidic linkage glycosylation sites are marked by double underlining.

[0042] The sequence of the human, processed (extracellularly) ActRIIa polypeptide is as follows:

TLGRSETQECLFFNANWEKDRTNQTCVEPCYGDKDKRRHCFATWKNISG SIEIVKOGCWLDDI.NCYDRTDCVEKKDSPEVYFCCCEGNNCNEKFSYFP EMEV_TQPTSNPVTPKPF (SIiQ ID end name: 2). The "tail" removed sequence (Δ15 sequence) is as follows:

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISG SIΕIV KOGCWL DDINC ¥ DRT DCVEKKDS PGV ¥ FCCCBGNMCNEK FS Ϊ FP EM <SEQ ID NO: 3) [0044] The nucleic acid sequence of the nucleotide, coding for the nucleotide, coding for the nucleotide, encoded with the sequence

ATGGG AG CTGC TGCAAAGTTG GCGT TTGCCGT CT TTCTTATCT CCTGTT CTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAG TęrCTTTTCTT TAATGC7AATTGGGAGAGAGAAGA C C AAT CAAACT GGTGTTGA

TGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTCCTACCTGGAAGA AT ATT TC TGGTT CC A TTGAAATAGT GAAACAAGG TTGTTGGCT GGńTGA

TATCAACTGCTATGńCAGGACTGATTGTGTAGAAAAAAAAGACAGCCCT GAAGTATATT TT TGTTGCT GT GAGGGCAATATG T GTAATGAAAAGTT TT CTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTAC

ACCTAAGCCACCCTATTACAACA7CCTGCTC TAT TCC TTG G TGCCACTT ATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATC ACAAGATGG CCT ACCCTCC T GTAC TTGTTC C AA CT CAAGA CCCTACAGAGCCCACCTA

GT GAAA GCAAG GGGAAGATTTGGT TGTGTCTGGAAAGCCCAG TT GC TTA

ACGAATATGTGGCTGTCAAAATATTTCACAACACAGGACAAACAGTCATG G CA AAA TGAAT AC GAA GTC TACĄGTT TGCC TGGAATG AAG CA TG AG AAC A TAT TACAGT TCATTGG TGCAGAAAAAC GAGGCA C CAGGGGTTATATT

T CT T AAG GCT AAT GT GG T CTC TT GGAA TG AA CTG TC TC AT ATTGC AC AA

A CC ATGGCTAGAG GA TTG GCATATT TACATGAGG AT ATACCTGGC CTAA AAG AT GGCCACAAACC TG CC AT ATC TC AC AG GG AC ATCAAAAG TAAAAA

TGTGCTGTTGAAAAACAACCTGflCAGCTTGCATTGCTGACTTTGGGTTG G CCTTAAAAT T TCAGGC TG GC AAGTCTGCAGGCGAT AC CCATGGACAGG TTGG ΓΑ C CCG GAGGTACATGGCTC CAGAG G TA TTAGAGGGTGCTATAAA CTT CC AAAGGGATGCAT TT TTGAGGATAGATATGTATGCCATGGGATTń GTCCTATGGGAACT GGC TTCTCGCTGTAC TGCT G CAGA TGGACCTCTAG A TG AATACATGT 'T GCCń TTTGAGGA GGAAAT TGGC C AGCATCC ATCTCT TG AAGACATG C AGG AAG TTGTT GTGCA TAĄAAAAAAGAGG CCTĆTTTTA AGAGATTATT GGCAGAAACATGC TGGAATGGC AATGC T CTGTGAAACCA TTGAAGAATGTTGGGATCAGGACGCAG AAGCC AGGTTArCAGCTGGńTG tgtaggtgaaagaattacccagatgcagagactaacaaatattattacc ACAGAG GAc ATT GT AAC AGT G GTCACAATGGTGACAAA TGT T GACTTTC CTCCCAAAGAATCTAGTCTATse (SEQ fD NO: sequence cod as sequence)

ATACTTGGTAG ATC AGAAACTCAGGAGTGTCT TTTCT T TAATG C ΓΑΑΤΤ GGGAAAAA G ACńG AACC AATC AAACTGGT AAC GTT G C C GTGT TATGGTGA AAAGATAAACGGCGG CATTGTTTTGCTACCTGGAAGAATATTT CTG GT TCCATTGńAATAGT GAAACAAGGT tgttggct gg at gatatcaac tgct ATG AC AGGACTGAT TGT GTAGAAAAAAAAG AC AGCCCTG AAGT AT ATTT TTGTTGCTGTGAGGGCAATńTGTGTAATGAAAAGT T TTCTTA? TT TCCA GAG AT GGAA GTCACACA GCOCACTTCAAAT CCAGTTACACC TAAGCCAC CC (SEQIDNO: 5) [0046] According to the description, the term "ActRIIb" refers to proteins of the type IIb activin receptor family (ActRIIb) from any species and variants derived from such ActRIIb proteins by mutagenesis or other modification. A reference to ActRIIb in the specification is understood to refer to any of the currently identified forms. ActRIIb family members are generally transmembrane proteins, composed of a ligand-binding extracellular domain, with a cysteine-rich region, transmembrane domain and cytoplasmic domain, with predicted serine / threonine kinase activity.

[0047] The term "ActRIIb polypeptide" includes polypeptides comprising any naturally absent polypeptide - a member of the ActRIIb family, as well as any variants thereof (including mutants, fragments, fusions and peptidomimetic forms) that retain useful activity. See, for example, WO / 2006/012627. For example, ActRIIb polypeptides include polypeptides derived from the sequence of any known * ActRIIb having a sequence at least about 80% identical to the sequence of the ActRIIb polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity. For example, an ActRIIb polypeptide of the invention may bind to and inhibit the function of an ActRIIb protein and / or activin. The ActRIIb polypeptide can be selected for activity in stimulating red blood cell formation in vivo. Examples of ActRIIb polypeptides include the human ActRIIb polypeptide precursor (SEQ ID NO: 15) and soluble human ActRIIb polypeptides (e.g., SEQ ID NOs: 16, 17, 20 and 21).

[0048] The sequence of the human ActRIIb precursor protein is as follows:

MTAPWALALLWGSLHFGS GRGEAJETPECIY TOANWZLE R 0) jQ S GLERC EXKRLHC YA $ WĄN) Ξ SGT Ϊ ĘŁVKrGCWIjDPF1JC YlJRQE CVATEENPQ VYFCCCE GNFCKE RFTLLPGLTLLPGLTLPLPG

A GLSLIVLLAFWMYRHRKPPYGHVD1HEDPGPPPPSPLVGLKPLQLLEIK RG R FGC VW KA Q LMN D FVAV K1F PLO DKQ Ξ WQ SER EI FST PGM KH FR LL QFTAAEKttGSNLEVELWLITAFHDKGSLrDYLKGh'I ITWNEI, CHVAETW SRGL5YLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKS [łLTAVLAUFGLA VRFEPGKPPGDT 4GQVGTRRYMAPEYLEGA1N FQRDAFLR1 DMYAMGLY LWELVSRGKAADGFVDEYMLPFEEElGQHPSLEELQEVWHKKMRPTIK 0H W LK H PG LAQLCVTIE ECW DH DAE A RLSAGC VE ER US L. IR RS VN GTT S DCL ™ SLVTSVTNVDLPPKESSI (SEQ ID NO: 15) [0049] The signal peptide is underlined once; the extracellular domain is in bold, and the potential N-linked glycosylation sites of glycosylation are boxed.

[0050] The sequence of the human, processed (extracellularly) ActRIIb polypeptide is as follows:

SGRGEAETRECI ¥ YNANWELERTNQSGLERCEGEQDKRLHCYASWANSS GTIELVKKGC WLDDFNCYDRQ EG VATEEN PQVY FCCC EGM FCN ERFTHL PEAGGPEVTYEPPPTAPT (SEQ ID NO: and 6) [0051] tail pos. The "tail" removed sequence (Δ15 sequence) is as follows:

SG RG E AET REG IY YN AN IN ELE RTNQSGLERC EGEQD KRLH CY AS W AN 5 S GTIELVKKGC HLDDFNC Y DRQECVATE EN FQVY FCCCEGN FCNERFT HL PEA (SEQ ID NO: 17) [0052] The nucleic acid sequence encoding the human protein as follows: (nucleotides 5-1543 of the entry Genbank NM_001106)

ATG AC GGCGCC CT G GG TGG CC CTCGCCCT CC TC TGGGGATCGCTGTGGC CCG GC TCTG GG CG T GG GG A GGĆTGAGACACGG GAGTGC ATC TAC TACAA CGCCAACTGGGAGCTGGCAGGGCCGCCCT

G AAGG CGAG CAGGACAA GC GGC TG CAC TGCTACG CCT C CTG GGC CAAC A GCT CT GGCA CC AT CG AGC T CG TGAAGAAG GG CT G CT G GCTAGAT GAGTT CAAC TGCTAC GATAGGCAGGAG TGTGTC-G CC AC TGC CC

GTG T AC TTCT GC TGC TG TGAAG GC AACTT C TGC AAC G AGC GCT TC AC TC

AT TTGC CAG AG GC T GGG G GC CC GG AAG TC ACGT AC GAG CC ACC CC CGAC

AGC CC CCAC CC TGCTCAC GGT GC ΓGGCCT ACTC ACTG C ​​TGC CC ATCGGG GGCCTTTCCCTCATCGTCCTGCTGGCCTTTTGGATGTACCGGCATCGCA

AGCC CCCCTA CGG TCAT GTGGACATCCA T GAGGAC C CT GG GCC TCCACC

ACCA T CCCC TC TG GT GG GCC T GAAGCCAC TGCAGC T GC TG GAG ATCAAG GC TC GGGGGCGC TTTGGCT GTGTCTGGAAGGCCC AGCT CA TGAAT GACT TT GTAGCTGT C AAGATC TTC CCAC TCCAGGACAAGCAG TC GTG GC AGG

T GAA CG GGAGAT CT TC AGC A CACC TGGCATGAAGCAC GAGAACCTGCTA

CAGTTCATTGCTGCCGAGAAGCCAGGCTCCAACCTCGAAGTAGAGCTGT

GGCTCATCACGGCCTrCCATGACAAGGGCTCCCTCACGCATTACCTCAA

GG GGAA CATCATC ACA TGG AAC GAACTG TGTCATG TAGCAGAGAC GATG TC AC GAGGCC TC T CATACC T GC AT GAGGA TGTGCCCTG GT GCCGT GG CG AG GG CC AC A AGC CCTCT AT 'Γ GC CC ACAC GG AC TTT A AAT GT A AAG AGCC-A CC TC AC AGC CGTG CT GG C TG ACTT TGGCT T CGCT G TTCG AT TT GAG CC AG GG AA AC CTC C AGG GG AC AC CC AC GG AC AGG TAG

GCAC GAGACG GTA CA TGGC T CC TG AGGT GC TCGAGGGA GCC AT CAACTT

C CAG AG AGA T GC CT TC CTG CGC AT T GACAT GT ATG CC ATGG GGT TGGT G CTGTGGGAGCTTGTGTCTCGCTGCAAGGCTGCAGACGGACCCGTGGATG

AG T ACATGCTGCCCTTTGAGG AAGAGATTGGCCAGC ACCCTTCGTTG GA G GA.GCTGCAG GAGGTGGTGGTGCACAAGAAGATGAGGCC CACCATTAAA GATCftĆTŚGTTGAAACACCCGGGCCTG GC CCAgcTtTGTGTC ACCATCC G AG AG GG ACC TGC TG ATGAT GCAG AGGCTCG C TTCTCCGCCGGCTGTGT ggaggagcgggtgtccctgattcggaggtcggtcaacggcactacctcg GACTGT C TCGTTTCCCTCGTGACC TCTGTCAC CAA TGTGGA CCTG CCCC CTAAAGAGTCAAGCATCTAA (3EQ ID NO: JS) [0053] The sequence of nucleic coding for a human soluble (extracellular) ActRIIa polypeptide is as follows:

TCTGGGCGTGGGGAGGC TGAG ACACGGGAGTGC AT CTACTACAAC GC CA ACTGGGAGCTGGAG CGCAC CAACCAGAGCGGCCTGGAGCGC TGCGAAG G CGAGCA GGACAAGC GGCTG CACTGCTACGCC TC CTGGGC CAACAG CTCT GGCACCATCGAGCTCGTGAAGAAGGGCrGCTGGCTAGATGACTTCAACT GCTACGATAGGCAG GAGTGTG TGGOCACTGAGGAGAACCCC C AGGTA CTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTG GTA CC AGAG GCTGGGGGCCC GGAAG TCACGTACGAGC C ACCCC CGACAGCC C CCACC (SEQ ID NO: 19) [0054] In a specific embodiment, the invention relates to the uses of certain soluble ActRII polypeptides. As used herein, the term "soluble ActRII polypeptide" generally refers to polypeptides comprising the extracellular domain of an Ac5 tRIIa or ActRIIb protein. The term "soluble ActRII polypeptide," as used herein, includes any naturally occurring extracellular domain of the ActRIIa or ActRIIb protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms). An activin-binding ActRII polypeptide is one that retains the ability to bind to activin, including, for example, AA, AB or BB activin or forms that contain the C or E subunit. Optionally, the activin-binding ActRII polypeptide will bind to activin AA with a dissociation constant of 1 nM or less. The extracellular domain of the ActRII protein binds to activin and is generally soluble, and therefore can be called a soluble, activin-binding ActRII polypeptide. Examples of soluble, activin-binding ActRII polypeptides include soluble polypeptides illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQ ID NO: 7 is referred to as ActRIIa-hFc, and is further described in the examples. Other examples of soluble, activin-binding ActRIIa polypeptides contain a signal sequence in addition to the extracellular domain of the ActRIIa protein, for example, the bee honey melitin leader sequence (SEQ ID NO: 8), the leader of tissue plasminogen activator (TPA) (SEQ ID NO: 9 ) or native Ac20 tRIIa leader (SEQ ID NO: 10). The ActRIIa-hFc polypeptide, illustrated in SEQ ID NO: 13, uses the TPA leader. Examples of soluble, activin-binding ActRIIb polypeptides include soluble polypeptides illustrated in SEQ ID NOs: 16, 17, 20. Activin-binding ActRIIb polypeptides may also contain a signal sequence, in addition to the extracellular domain of the ActRIIb protein, for example, the myelapine melitin leader sequence SEQ ID NO: 8), or the leader of tissue plasminogen activator (TPA) (SEQ ID NO: 9).

[0055] Functionally active fragments of ActRII polypeptides can be obtained by screening recombinantly produced polypeptides from the corresponding nucleic acid fragments encoding the ActRII polypeptide. In addition, fragments can be chemically synthesized using techniques known in the art, such as conventional solid phase Merrifield chemistry, f-Moc or t-Boc. Fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can act as antagonists (inhibitors) of ActRII protein or activin mediated signaling.

[0056] Functionally active fragments of ActRII polypeptides can be obtained by screening libraries of modified polypeptides, recombinantly produced from the corresponding mutagenized nucleic acids encoding the ActRII polypeptide. Variants can be produced and tested to identify those that may act as antagonists (inhibitors) of ActRII protein or activin mediated signaling. The functional variant of ActRIIa polypeptides may contain an amino acid sequence that is at least 75% identical to the amino acid sequence selected from SEQ ID NOs: 2 or 3. In some cases, the functional variant has an amino acid sequence at least 80%, 85%, 90% , 95%, 97%, 98%,

99% or 100% identical to the amino acid sequence selected from SEQ ID NOs: 2 or 3. The functional variant ActRIIb polypeptides may contain an amino acid sequence that is at least 75% identical to the amino acid sequence selected from SEQ ID NOs: 16 or 17. In in some cases, the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence selected from SEQ ID NOs: 17 or 18.

[0057] Functional variants can be generated by modifying the structure of an ActRII polypeptide for purposes such as enhancing therapeutic efficacy or stability (e.g., allowed ex vivo storage life and resistance to in vivo proteolytic degradation). Such modified ActRII polypeptides, when selected with preservation of activin binding, are considered functionally equivalent to naturally occurring ActRII polypeptides. Modified ActRII polypeptides can also be produced, for example, by amino acid substitution, deletion or addition. For example, it is rational to expect that the isolated conversion of leucine to isoleucine or valine, aspartate to glutamate, threonine to serine or a similar amino acid conversion to a structurally related amino acid (e.g. conservative mutation) will not have a significant effect on the biological activity of the resulting molecule. Conservative replacements are those that occur within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a given ActRII polypeptide will result in a functional homolog can be easily determined by assessing the ability of a variant ActRII polypeptide to produce a response in cells in a manner similar to a wild-type ActRII polypeptide.

[0058] In some embodiments, the disclosure contemplates specific mutations of ActRII polypeptides leading to a change in glycosylation of the polypeptide. Such mutations can be selected to introduce or eliminate one or more glycosylation sites, such as O- or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally contain a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid) that is specifically recognized by the appropriate cellular glycosylation enzymes. Changes can also be made by the addition or substitution of one or more serine or threonine residues to the wild-type ActRII polypeptide sequence (to O-glycosidic linkage glycosylation sites). A series of amino acid substitutions or deletions at one or both of the first or third amino acid positions of the glycosylation recognition site (and / or an amino acid deletion at the second position) results in a lack of glycosylation in the modified tripeptide sequence. Another way to increase the number of carbohydrate moieties on an ActRII polypeptide is by chemical or enzymatic coupling of glycosides to the ActRII polypeptide. Depending on the conjugation model used, the sugar (s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free thiol groups such as these cysteines; (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the glutamine amide group. Removal of one or more carbohydrate moieties present on an ActRII polypeptide can be carried out chemically and / or enzymatically. Chemical deglycosylation may require, for example, exposure of the ActRII polypeptide to a trifluoromethanesulfonic acid compound or equivalent compound. This treatment results in the cleavage of most or all sugars, except for binding sugar (N-acetylglucosamine or N-acetylgalactosamine), while the amino acid sequence is left intact. The enzymatic cleavage of carbohydrate moieties on ActRII polypeptides can be achieved by using a number of endo- and exo-glycosidases, as described in Thotakura et al. (1987) Meth. Enzymol. 138: 350. The ActRII polypeptide sequence can be adjusted as needed depending on the type of expression system used, as mammalian, yeast, insect and plant cells can all introduce different glycosylation patterns that can be affected by the peptide amino acid sequence. In general, ActRII proteins for human use will be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other expressed mammalian cell lines are expected to be equally useful.

[0059] The specification further contemplates the method of generating mutants, particularly sets of combinatorial mutants of an ActRII polypeptide, as well as truncated mutants; pools of combinatorial mutants are particularly useful for identifying functional sequence variants. The purpose of performing such combinatorial library screening may be to generate, for example, variants of an ActRII polypeptide that bind to activin or other ligands. A number of screening assays are provided below, and such assays can be used to evaluate variants. For example, an ActRII polypeptide variant may be screened for its ability to bind ActRII ligand, prevent binding of ActRII ligand to ActRII polypeptide, or interfere with ActRII ligand-induced signaling.

[0060] The activity of an ActRII polypeptide or variants thereof can also be tested in a cellular assay or in vivo. For example, the effect of an ActRII polypeptide variant on the expression of genes involved in hematopoiesis can be assessed. This can be done as required in the presence of one or more recombinant ActRII ligand proteins (e.g., activin), and the cells can be transfected to produce an ActRII polypeptide and / or variants thereof, and optionally, an ActRII ligand. Similarly, the ActRII polypeptide can be administered to mice or another animal, and one or more blood measurements, such as RBC number, hemoglobin concentration, or reticulocyte number can be assessed.

[0061] Generation of combinatorial variants that have selective or generally increased potency over naturally occurring ActRII polypeptide can be generated. Similarly, mutagenesis can lead to variants that have intracellular half-lives dramatically different from the corresponding polypeptide.

Wild-type ActRII. For example, the altered protein can be made more stable or less stable against proteolytic degradation or other cellular processes that lead to the degradation or inactivation of another type of native ActRII polypeptide. Such variants, and the genes encoding them, can be used to alter ActRII polypeptide levels by modulating the half-life of ActRII polypeptides. For example, a short half-life may lead to more transient biological effects and allow tighter control of recombinant ActRII polypeptide levels in the cell. In an Fc fusion protein, mutations can be introduced at the linker (if any) and / or a portion of Fc to change the half-life of the protein.

[0062] A combinatorial library can be created through a degenerate library of genes encoding a library of polypeptides, each of which contains at least a portion of the potential ActRII polypeptide sequences. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence such that a degenerate set of potential ActRII polypeptide sequences are expressed as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage presentation).

[0063] There are many ways to generate a library of potential homologues from a degenerate oligonucleotide sequence. Chemical synthesis of the degenerate gene sequence can be performed in an automated DNA synthesizer, and the synthetic genes are then ligated into a suitable expression vector. 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). Such techniques have 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; as well as US Patent Nos. 5,223,409, 5,198,346 and 5,096,815).

[0064] Alternatively, other types of mutagenesis can be used to generate a combinatorial library. For example, variants of ActRII polypeptides can be generated and isolated from the library by screening using, for example, alanine scanning mutagenesis and the like (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), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193: 653-660; Brown et al., (1992) Mol. Cell Biol. 12: 26442652; McKnight et al., (1982) Science 232: 316); by saturation mutagenesis (Meyers et al., (1986) Science 232: 613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1: 11-19); or random mutagenesis, including chemical mutagenesis, etc. (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). Linkage scanning mutagenesis, especially in a combinatorial system, is an attractive way to identify truncated (bioactive) forms of ActRII polypeptides.

[0065] A wide range of techniques are known in the art for screening gene products from combinatorial libraries obtained by point mutations and truncations, and, as discussed, for screening cDNA libraries for gene products having a specific property. Such techniques will generally be adaptable for rapid screening of gene libraries generated by combinatorial mutagenesis of ActRII polypeptides. The most commonly used techniques for screening large gene libraries typically include cloning the gene library into replication-capable expression vectors, transforming the respective cells with the resulting library of vectors, and expressing combinatorial genes under conditions in which detection of desired activity is facilitated by relatively simple isolation of the vector encoding the gene whose product detected. Preferred assays include activin binding assays and activin-mediated cell signaling assays.

[0066] In some embodiments, ActRII polypeptides useful according to the invention may additionally contain post-translational modifications, in addition to those that are naturally present in ActRII polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. As a result, modified ActRII polypeptides may contain non-amino acid elements such as polyethylene glycols, lipids, poly- or mono-saccharides and phosphates. The effects of such non-acid elements on the functionality of an ActRII polypeptide can be tested as described for other ActRII polypeptide variants. When an ActRII polypeptide is produced in cells by cleaving the native form of the ActRII polypeptide, post-translational processing may also be important for proper folding and / or protein function. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities, and can be selected to ensure correct modification and processing of ActRII polypeptides.

[0067] Functional variants or modified forms of ActRII polypeptides may contain fusion proteins having at least a portion of ActRII polypeptides and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, 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 chosen to confer the desired property. For example, some fusion domains are particularly useful for isolating fusion proteins by affinity chromatography. For affinity-based purification, appropriate affinity chromatography matrices such as glutathione, amylase and nickel or co-conjugate resins are used. Many such matrices are available in the form of a "kit" such as the Pharmacia GST purification system and the QIAexpress ™ system (Qiagen) useful with fusion partners (HIS6). As another example, the fusion domain can be chosen to facilitate detection of ActRII polypeptides. Examples of such detection domains include various fluorescent proteins (e.g., GFP) as well as "epitope markers", which are typically short peptide sequences for which a specific antibody is available. Well known epitope markers for which specific monoclonal antibodies are readily available include FLAG markers, influenza hemagglutinin (HA) and c-myc markers. In some cases, the fusion domains have a cleavage site for a preotease, such as for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby release recombinant proteins from them. The released proteins can then be isolated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, the ActRII polypeptide may be conjugated to a domain that stabilizes the ActRIIa polypeptide in vivo ("stabilizer" domain). By "stabilizing" is meant any action that extends the serum half-life, regardless of whether this is due to reduced destruction, reduced renal clearance, or another pharmacokinetic effect. Fusions with the Fc portion of immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Similarly, fusions with human serum albumin may confer desirable properties. Other types of fusion domains that can be selected include multimerization (e.g., dimerization, tetramerization) of domains and functional domains (which confer additional biological function, such as further stimulation of muscle mass growth).

[0068] As a specific example, the invention uses a fusion protein comprising a soluble ActRIIa extracellular domain conjugated to an Fc domain (e.g.

SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFFFKPKDTLMISRTPBVTCWVD f A) VSH EDPEVKFNWYVOG VEVHNAKTKPREŁQΥ Ν ΞTYRVVSVLT7LJJQDWLNGKEYKCK (A} USNKALF V PIΞKTIΞKAK GQ PRE PQVYTL PPS ftEEMT KNQVS LTCLV KG FY PS DIAVEWE SNGQ PE NH Ϊ KT Γ? P VL DS DG P FFL YS KLT V OKS RWQOGN VF sc S VMH EALH N [0069] As an additional specific example, the invention uses a fusion protein comprising a soluble ActRIIb extracellular domain conjugated to an Fc domain (eg, SEQ ID NO: 21).

SGW3EACTRECTYYMANMELERTNQSGLEACEGE0DiKRLHCVAEHAMSSGTlELVKKGCWLE>

D FNCΪ DRQ ECVATEEN PQVYFCCCEGU FCNER FTHLPE AGG PEVT t E PP ΡΓΑPTGGGT HT CP PC PAPĘLLGG PS 7FLF PPK PK DTLMISRT PEVTC WVDVSH E DP EV KΪΉ WYVDGVΞVH HA KT KPREEQYWSTLPKKKKQKKQKK

ΡPSREEMTKMQVSLTCLVKGFYPSDIAV £ WESNGQΡΕΝΝΪΚΤΤPPVLD5DGSFFLΪSKLTVD KS RWQQGN VFSC Ξ VMΗ EAL JIH Fi YTQKS LE LE PGK [0070] Optionally, the Fc domain has one or more mutations of 32 residues, such as Asp2, 4 residues, such as Asps. In some cases, an Fc domain mutant having one or more of these mutations (e.g., an Asp-265 mutation) has reduced ability to bind to Fcy receptors relative to the wild type Fc domain. In other cases, an Fc domain mutant having one or more of these mutations (e.g. Asn-434 mutation) has increased binding capacity to the Fc receptor associated with MHC class I (FcRN) relative to the wild-type Fc domain.

[0071] It is understood that the various elements of the fusion proteins can be arranged in any manner that is compatible with the desired functionality. For example, the ActRII polypeptide may be C-terminal to the heterologous domain, or, alternatively, the heterologous domain may be C-terminal to the ActRII polypeptide. The ActRII polypeptide domain and heterologous domain need not be adjacent to the fusion protein, and additional domains or amino acid sequences, C- or N-terminally relative to any of the domains, or between domains may be present.

[0072] In some embodiments, ActRII polypeptides useful according to the invention comprise one or more modifications that are capable of stabilizing ActRII polypeptides. For example, such modifications increase the in vitro half-life of ActRII polypeptides, increase the circulating half-life of ActRII polypeptides, or reduce the proteolytic degradation of ActRII polypeptides. Such stabilizing modifications include, but are not limited to; fusion proteins (including, for example, fusion proteins containing an ActRII polypeptide and stabilizer domain), glycosylation site modifications (including, for example, glycosylation site addition to an ActRII polypeptide) and carbohydrate moieties (including, for example, deletion of moieties carbohydrate from an ActRII polypeptide). As used herein, the term "stabilizer domain" does not only apply to the fusion domain (e.g. Fc) as for fusion proteins, but also includes non-protein modifications, such as a carbohydrate moiety, or a non-protein moiety, such as polyethylene glycol.

[0073] In some embodiments, the invention makes available and / or purified forms of ActRII polypeptides that are isolated from or otherwise substantially released from other proteins. ActRII polypeptides will generally be expressed by recombinant nucleic acids.

3. Nucleic Acids Encoding ActRII Polypeptides [0074] We disclose isolated and / or recombinant nucleic acids encoding any ActRII polypeptides (e.g., soluble ActRIIa polypeptides and soluble ActRIIb polypeptides), including fragments, functional variants and fusion proteins disclosed herein. For example, SEQ ID NO: 4 encodes the naturally occurring human ActRIIa precursor polypeptide, while SEQ ID NO: 5 encodes the extracellular domain of ActRIIa after processing. For example, SEQ ID NO: 18 encodes the naturally occurring human ActRIIb precursor polypeptide, while SEQ ID NO: 19 encodes the extracellular domain of ActRIIb after processing. The subject nucleic acids can be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids can be used, for example, in methods for producing ActRII polypeptides or as direct therapeutic agents (e.g., in a gene therapy approach).

[0075] In some aspects, the subject nucleic acids encoding ActRIIa polypeptides are further understood to include nucleic acids, which are variants of SEQ ID NO: 4 or 5. In some aspects, the subject nucleic acids encoding ActRIIb polypeptides are further understood to include nucleic acids, which are the variants of SEQ ID NO: 18 or 19. Variant nucleotide sequences include sequences that differ in one or more nucleotide substitutions, additions or deletions, such as allelic variants.

[0076] Isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4, 5 are disclosed. 18 or 19. The skilled artisan will recognize that nucleic acid sequences complementary to SEQ ID NO: 4, 5, 18, or 19 can be provided, and SEQ ID NOs: 4, 5, 18 or 19 variants. Nucleic acid sequences can be isolated, recombinant sequences and / or linked to a heterologous nucleotide sequence, or in a DNA library.

[0077] Nucleic acids may also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 4, 5, 18, or 19, the complementary sequences of SEQ ID NO: 4, 5, 18 or 19, or fragments thereof. As discussed above, one of ordinary skill in the art will readily understand that stringent conditions that promote DNA hybridization may vary. One skilled in the art will readily understand that stringent conditions that promote DNA hybridization may vary. For example, hybridization in 6.0 x sodium chloride / sodium citrate (SSC) can be carried out at about 45 ° C, followed by a wash with 2.0 x SSC at 50 ° C. For example, the salt concentration at the washing step can be selected from low stringency of about 2.0 x SSC at 50 ° C to high stringency of about 0.2 x SSC at 50 ° C. In addition, the temperature at the washing step can be increased from low stringency conditions at room temperature, about 22 ° C, to high stringency conditions at about 65 ° C. Both temperature and salt can be varied, or temperature or salt concentration can be kept constant while another variable changes. It is possible to provide nucleic acids that hybridize under low stringency conditions, 6 x SSC at room temperature, followed by 2 x SSC wash at room temperature. [0078] Isolated nucleic acids that differ from nucleic acids as set out in SEQ ID NOs: 4, 5, 18 or 19 due to the degeneracy of the genetic code are also possible to provide. For example, a number of amino acids are encoded by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) can lead to "silent" mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that lead to changes in the amino acid sequences of the subject proteins are expected to occur between mammalian cells. One skilled in the art will recognize that these variations in one or more nucleotides (up to about 3-5% nucleotides) of nucleic acids encoding a particular protein may occur in individual individuals of a given species due to natural allelic variation.

[0079] Recombinant nucleic acids may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of suitable expression vectors and appropriate regulatory sequences are known in the art for a number of host cells. Typically, one or more nucleotide regulatory sequences may contain, without limitation, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated. Promoters can be either naturally occurring promoters or hybrid promoters that combine elements of more than one promoter. The expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be introduced into the chromosome. The expression vector may contain selectable marker genes to allow selection of transformed host cells. The selectable marker genes are well known in the art and will vary depending on the host cells used.

[0080] The nucleic acid may be provided in an expression vector comprising a nucleotide sequence encoding an ActRII polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are known in the art, and selected to drive expression of the ActRII polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide range of expression control sequences that control the expression of a DNA sequence when operably linked thereto can be used in these vectors to express the DNA sequence encoding the ActRII polypeptide. Such useful expression control sequences include, for example, SV40 early and late promoters, tet promoter, adenovirus or cytomegalovirus direct early promoter, RSV promoters, lac system, trp system, TAC or TRC system, T7 promoter directed by T7 RNA polymerase , major operator and promoter regions of lambda phage, fd coat protein control regions, promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters, e.g. Pho5, promoters of yeast α conjugation factors, the polyhedron promoter from the baculovirus system, and other sequences known to control the expression of prokaryotic or eukaryotic cell genes or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of host cell for transformation and / or the type of protein that expression is desired. Furthermore, the copy number of the vector, the ability to control that copy number, and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. [0081] The recombinant nucleic acid can be produced by ligating the cloned gene or part thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammal), or both. Expression carriers for the production of a recombinant ActRII polypeptide include plasmids and other vectors. For example, suitable vectors include plasmids of pBR322 derived plasmids, pEMBL derived plasmids, pEX derived plasmids, pBTac derived plasmids and pUC derived plasmids for expression in prokaryotic cells such as E. coli.

[0082] Some mammalian expression vectors contain both prokaryotic sequences to facilitate the multiplication of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from peDNAI / amp, peDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug-based selection in both prokaryotic and eukaryotic cells. Alternatively, virus derivatives, such as bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, derived from pREP and p205) can be used to transiently express proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems. Various methods used in plasmid preparation and transformation of host organisms are well known in the art. Other suitable expression systems for both prokaryotic and eukaryotic cells as well as general recombination procedures, see Molecular Cloning A Laboratory Manual, 3rd edition, ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393 and pVL941), vectors derived from pAcUW (such as pAcUW1), and vectors derived from pBlueBac (such as pBlueBac III containing β-gal).

[0083] A vector can be designed for producing the ActRII polypeptides in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), PcDNA4 vectors (Invitrogen, Carlsbad, Calif.) And pCl-neo vectors (Promega, Madison , Wisc.). As will be obvious, the subject gene constructs can be used to elicit expression of the subject ActRII polypeptides in culture-grown cells, e.g., for the production of proteins, including fusion proteins or protein variants, for purification.

[0084] We disclose herein a host cell transfected with a recombinant gene, including a coding sequence (e.g., SEQ ID NO: 4, 5, 18 or 19) for one or more of the ActRII polypeptides of interest. The host cell may be any prokaryotic or eukaryotic cell. For example, the ActRII polypeptide of the invention can be expressed in bacterial cells, such as E. coli, insect cells (e.g. using a baculovir expression system), yeast or mammalian cells. Other suitable host cells are known to those of skill in the art.

[0085] Accordingly, disclosed herein are methods for producing the ActRII polypeptides of interest. For example, a host cell transfected with an expression vector encoding an ActRIIa or ActRIIb polypeptide may be cultured under appropriate conditions to allow expression of the ActRII polypeptide to occur. The ActRII polypeptide may be secreted and isolated from a mixture of cells and medium containing the ActRII polypeptide. Alternatively, the ActRII polypeptide can be preserved in the cytoplasm or membrane fraction and harvested cells, lyzed and protein isolated. The cell culture includes host cells, media and other by-products. Suitable media for cell cultures are well known in the art. The subject ActRIIa polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for protein purification, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification, with specific antibodies for specific epitopes of ActRII polypeptides and immunoaffinity purification, with agent, which binds to an ActRIIa-conjugated domain (e.g., a protein A column can be used to purify an ActRIIa-Fc or ActRIIb-Fc fusion). The ActRII polypeptide may be a fusion protein containing a domain that facilitates its purification. Purification can be accomplished by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be completed by virus filtration and buffer exchange. As demonstrated herein, the ActRIIa-hFc protein was purified to> 98% purity as determined by size exclusion chromatography and> 95% as determined by SDS PAGE. This level of purification was sufficient to achieve the desired results in mice, rats and non-human primates.

[0086] A fusion gene encoding a purified leader sequence, such as a poly (His) / enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant polypeptide

ActRII, may enable purification of the expressed fusion protein by chroma2 + affinity chromatography using a Ni metal resin²⁺. The purified leader sequence can then be removed, by treatment with enterokinase, to provide the purified ActRII polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88: 8972).

[0087] Methods for producing fusion genes are known. Essentially, combining different DNA fragments encoding the sequences of different polypeptides is carried out according to conventional techniques, using blunt or protruding ends for ligation, restriction enzyme digestion to ensure the appropriate ends, filling the sticky ends as appropriate, alkaline phosphatase treatment to avoid unwanted combination and enzymatic ligation. The fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that lead to complementary protruding ends between two consecutive gene fragments that can later be hybridized to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

4. Screening Assays In some aspects, the invention provides a method of identifying an agent that increases red blood cell levels and relates to the use of ActRII polypeptides (e.g., soluble ActRIIa or ActRIIb polypeptides) and activin polypeptides to identify compounds (agents) that are agonists or antagonists of the pathway signaling activin-ActRIIa and / or activin-ActRIIb. Compounds identified through these screening tests can be tested to assess their ability to modulate red blood cell, hemoglobin and / or reticulocyte levels in vivo or in vitro. These compounds can be tested, for example, in animal models.

[0089] There are numerous approaches to screening for therapeutic agents for increasing red blood cell or hemoglobin levels by targeting activin and ActRII signaling. In some embodiments, high-throughput compound screening can be performed to identify agents that interfere with the action of activin or ActRII on a selected cell line. In some embodiments, the assay is performed to screen and identify compounds that specifically inhibit or reduce binding of an ActRIIa or ActRIIb polypeptide to activin. Alternatively, the assay can be used to identify compounds that enhance the binding of an ActRIIa or ActRIIb polypeptide to activin. In a further embodiment, the compounds can be identified by their ability to interact with an activin, ActRIIb polypeptide or ActRIIa polypeptide.

[0090] A number of signage formats will be sufficient, and, in light of the disclosure, those not explicitly described herein will still be understood by one of ordinary skill in the art. As described, test compounds (agents) can be formed in any combinatorial chemical manner. Alternatively, the subject compounds may be naturally occurring biomolecules synthesized in vivo or in vitro. Compounds (agents) can be produced for testing for their ability to act as tissue growth modulators, for example, in bacteria, yeast, plants or other organisms (e.g., natural products), chemically produced (e.g., small molecules, including peptidomimetics) , or produce recombinantly. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones and nucleic acid molecules. In a specific embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2000 Daltons.

[0091] Test compounds of the invention may be provided as single, separate entities, or provided in libraries of greater complexity, such as those obtained by combinatorial chemistry. These libraries may contain, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Presentation of test compounds in the test system may occur either in isolated form or as a mixture of compounds, especially at the initial stages of screening. Optionally, the compounds may optionally be derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photoactivable crosslinkers or any combination thereof.

[0092] In many drug screening programs that test compound libraries and natural extracts, high throughput assays are desirable to maximize the number of compounds analyzed over a given period of time. Often, assays performed on extracellular systems, such as can be obtained from purified or partially purified proteins, as "primary" screening tests are preferred in that they can be generated to allow rapid development and relatively simple detection of molecular target change mediated by test compound. Furthermore, the effects of cellular toxicity or bioavailability of the test compound can generally be neglected in an in vitro system, instead the assay focuses mainly on the drug's effect on the molecular target, which can be manifested in a change in binding affinity between the ActRIIa polypeptide and activin and / or between the ActRIIb polypeptide and activin.

[0093] To illustrate, in exemplary screening assays of the invention, the subject compound is contacted with an isolated and purified ActRIIa polypeptide that is typically capable of binding to activin. A composition containing the ActRIIa ligand is then added to the mixture of compound and ActRIIa polypeptide. Detection and quantification of ActRIIa / activin complexes provides a method of determining the effectiveness of a compound in inhibiting (or enhancing) the formation of complexes between an ActRIIa polypeptide and activin. The efficacy of a compound can be assessed by generating dose response curves from data obtained using different concentrations of test compounds. Furthermore, a control determination can also be made to provide baseline for comparison. For example, in a control assay, isolated and purified activin is added to the composition containing the ActRIIa polypeptide, and formation of the ActRIIa / activin complex is quantified in the absence of test compound. It will be understood that, in general, the order in which reagents can be mixed can be varied, and they can be mixed simultaneously. Moreover, instead of purified proteins, cell extracts and lysates can be used to obtain the appropriate extracellular assay system. Compounds that affect ActRIIb signaling can be identified in a similar manner using an ActRIIb polypeptide and an ActRIIb ligand.

[0094] Complex formation between the ActRII polypeptide and activin can be detected by a variety of techniques. For example, modulation of complex formation can be quantified using, for example, detectable labeled proteins,

35 Such as radiolabelled (e.g. P, S, C or H), fluorescently labeled (e.g. FITC), or enzymatically labeled ActRIIa or ActRIIb polypeptide or activin, by immunoassay, or chromatographic detection.

[0095] In some embodiments, the invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer assays (FRETs) in measuring, direct or indirect, the degree of interaction between an ActRII polypeptide and its protein, a binding partner. In addition, other detection modes, such as those based on optical fibers (PCT publication WO 96/26432 and US Patent No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors and surface force sensors are compatible with many embodiments of the invention.

[0096] In addition, the invention contemplates the use of a trap-type interaction assay, also known as a "two-hybrid assay", to identify agents that interfere with or enhance the interaction between an ActRII polypeptide and its binding partner protein. See, for example, US 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 a specific embodiment, the invention contemplates the use of reverse hybrid systems to identify compounds (e.g., small molecules or peptides) that remove interactions between an ActRII polypeptide and its binding partner protein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res 27: 919-29; Vidal and Legrain, (1999) Trends Biotechnol 17: 374-81; and US Patent Nos. 5,525,490; 5,955,280; and 5,965,368.

[0097] In some embodiments, the subject compounds are identified by their ability to interact with an ActRII polypeptide or activin polypeptide of the invention. Interactions between the compound and an ActRIIa, ActRIIb or activin polypeptide may be covalent or non-covalent. For example, such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1). In some cases, the compounds can be screened using a mechanism-based assay, such as an assay for detecting compounds that bind to activin or ActRII polypeptide. This may include a solid phase or liquid phase binding event. Alternatively, the gene encoding the activin or ActRII polypeptide can be transfected in a reporter system (e.g., β-galactosidase, luciferase, or green fluorescent protein) into the cell and screened for the library, optionally by high-throughput screening or individual library members. Other assays based on binding mechanism may be used, for example, binding assays that detect changes in free energy. Binding assays can be performed with a target bound to a well, bead or chip, or captured by an immobilized antibody or separated by capillary electrophoresis. Bound compounds can usually be detected using colorimetry or fluorescence or surface plasmon resonance.

5. Therapeutic uses [0098] Activin-ActRII antagonists (e.g., ActRIIa or ActRIIb polypeptides) can be used to increase red blood cell levels in mammals such as rodents and primates, and particularly in human patients. Some activin-ActRII antagonists may be used in methods of treating or preventing anemia in a subject in need thereof, or in methods of stimulating red blood cell formation in a subject. These methods can be used in the therapeutic and prophylactic treatment of mammals, especially humans.

[0099] As used herein, a therapeutic that "prevents" or represents a compound that, in a statistical sample, reduces the incidence of the disorder or condition in the treated sample relative to the untreated control, or delays the onset or reduces the severity of one or more symptoms of the disorder. or condition, relative to the untreated control. The term "treatment" as described herein includes prophylaxis of a particular condition or improvement or elimination of a condition after it has stabilized. In each case, prevention or treatment can be distinguished in the diagnosis provided by the physician or other healthcare professional and the intended outcome of the therapeutic agent.

[0100] As shown herein, activin-ActRIIa antagonists and activinActRIIb antagonists can be used to increase levels of red blood cells, hemoglobin, or reticulocytes in healthy individuals, and such antagonists can be used in selected patient populations. Examples of suitable patient populations include those with undesirably low levels of red blood cells or hemoglobin, such as patients with anemia, and those who are at risk of developing undesirably low levels of red blood cells or hemoglobin, such as those who are about to undergo serious surgery or other procedures that can result in significant blood loss. A patient with an adequate level of red blood cells can be treated with an activin-ActRIIa antagonist or an activin-ActRIIb antagonist to increase red blood cell levels, followed by blood collection and storage for later use in transfusion.

[0101] The activin-ActRII antagonists disclosed herein, and especially the ActRIIa-Fc and ActRIIb proteins, can be used to increase red blood cell levels in anemic patients. When observing hemoglobin levels in humans, sub-normal levels for relevant age and sex categories may indicate anemia, although individual differences should be considered. For example, hemoglobin levels of 12 g / dL are generally considered the lower limit of normal in the general adult population. Potential causes include blood loss, nutritional deficiencies, drug reactions, various bone marrow problems and many diseases. More specifically, anemia may be associated with a number of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis and bone marrow transplantation. Anemia can also be associated with the following conditions: solid tumors (e.g. breast cancer, lung cancer, colorectal cancer); lymphatic neoplasms (e.g., chronic lymphocytic leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma); hematologic malignancies (e.g. leukemia, myelodysplastic syndrome, multiple myeloma); radiotherapy; chemotherapy (e.g. schemes containing platinum); inflammatory and autoimmune diseases including, but not limited to, rheumatoid arthritis, other arthritis, systemic lupus erythematosus (SLE), acute or chronic skin diseases (e.g. psoriasis), inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis); acute or chronic kidney disease or failure, including idiopathic or congenital conditions; acute or chronic liver disease; acute or chronic bleeding; situations where red blood cell transfusion is not possible due to patient allo- or auto-antibodies and / or for religious reasons (e.g., some Jehovah's Witnesses); infections (e.g. malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell disease, thalassemia; drug use or abuse, e.g. alcohol abuse; pediatric patients with anemia caused by any cause to avoid transfusion; and elderly or patients with underlying coronary artery disease and anemia who cannot receive transfusions due to concerns about cardiovascular overload. [0102] Patients can be treated with a dosage regimen to restore the patient's target hemoglobin, typically between about 10 g / dl and about 12.5 g / dl, and typically about 11.0 g / dl (see also Jacobs et al. (2000) Nephrol Dial Transplant 15, 15-19), although lower target levels may cause fewer cardiovascular side effects. Alternatively, hematocrit levels (percentage of the volume of blood sample occupied by cells) can be used as a measure of the state of red blood cells. Hematocrit levels in healthy individuals range from 41 to 51% for adult men and 35 to 45% for adult women. Target hematocrit levels are usually around 30-33%. In addition, hemoglobin / hematocrit levels vary from person to person. In this way, optimally, the target hemoglobin / hematocrit level can be individualized for each patient. [0103] The rapid interaction of the activin-ActRIIa antagonists disclosed herein on red blood cell levels indicates that these agents act through a mechanism other than Epo. Accordingly, these antagonists may be useful for increasing red blood cell and hemoglobin levels in patients who do not respond well to Epo. For example, the activin-ActRIIa antagonist may be beneficial for a patient in whom administration of a normal to elevated (> 300 IU / kg / week) dose of Epo does not result in an increase in hemoglobin to the target level. Patients with an inadequate Epo response can be found in all types of anemia, but higher numbers of non-responders have been seen particularly often in cancer patients and patients with end-stage renal disease. An inadequate response to Epo can be either constitutive (i.e., visible after the first Epo treatment) or acquired (e.g., visible after subsequent Epo treatments).

[0104] Activin-ActRII antagonists can also be used to treat patients who are susceptible to Epo side effects. Primary Epo side effects are excessive hematocrit or hemoglobin levels and polycythaemia. Elevated hematocrit levels can lead to hypertension (more specifically, worsening of hypertension) and vascular thrombosis. Other Epo side effects that have been reported, some of which are associated with hypertension, are headache, flu-like syndrome, fistula obstruction, myocardial infarction and cerebral convulsions, caused by thrombosis, hypertensive encephalopathy and red blood cell aplasia (Singibarti, (1994) J. Clin Investig 72 (suppl 6), S36-S43; Horl et al. (2000) Nephrol Dial Transplant 15 (suppl 4), 5156; Delanty et al. (1997) Neurology 49, 686-689; Bunn (2002) N Engl J Med 346 (7), 522523).

6. Pharmaceutical compositions [0105] Activin-ActRII antagonists (e.g., ActRIIa and ActRIIb polypeptides) can be formulated with a pharmaceutically acceptable carrier. For example, the ActRII polypeptide may be administered alone or as part of a pharmaceutical formulation (therapeutic composition). The subject compounds may be formulated for administration by any convenient route for use in medicine or veterinary medicine.

[0106] In some embodiments, the invention comprises administering the composition systemically, or topically, as an implant or device. After administration, the therapeutic composition for use according to the invention means, of course, a pyrogen-free, physiologically acceptable form. Therapeutically useful agents, other than ActRII antagonists, which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds (e.g. with ActRIIa and ActRIIb) polypeptides in the methods of the invention.

[0107] Typically, activin-ActRII antagonists will be administered parenterally. Pharmaceutical compositions suitable for parenteral administration may contain one or more ActRII polypeptides, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions. before use, which may contain antioxidants, buffers, b actinostatin, solutes, which make the formulation isotonic with the blood of the intended donor or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils such as olive oil and injected organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, the maintenance of the correct particle size in the case of dispersions, and by the use of surfactants.

[0108] In addition, the composition may be encapsulated or injected in a form intended to be delivered to the site of the target tissue (eg bone marrow). In some embodiments, the compositions of the invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., ActRIIa or ActRIIb polypeptides) to a target tissue site (e.g., bone marrow), providing a structure for tissue development and optimally capable of being absorbed by the body . For example, the matrix can provide slow release of ActRII polypeptides. Such matrices can be created from materials used in other medical implant applications.

[0109] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interfacial properties. The specific use of the subject compositions will define the appropriate formulation. Potential matrices for the compositions can be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermatological collagen. Further arrays consist of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined such as sintered hydroxyapatite, bioglass, clay or other ceramic materials. The matrices may consist of a combination of any of the above types of materials, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramic materials can be changed in the composition, as in calcium aluminum phosphate, and treated to change the pore size, particle size, particle shape and biodegradability.

[0110] In some embodiments, the compositions of the invention may be administered orally, e.g. in the form of capsules, sachets, pills, tablets, lozenges (using an aromatized base, usually sucrose and acacia or tragus), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water emulsion or water in oil, or as an elixir or syrup, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and acacia) and / or mouthwash and the like, each containing a predetermined quantity of agent as the active ingredient. The agent can also be given as an intravenous injection, medicine mixed with honey or syrup or paste.

[0111] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), one or more therapeutic compounds of the invention may be mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or admixtures, such as starches, lactose, sucrose, glucose, mannitol and / or silica; (2) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) moisturizing substances such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard gelatin capsules using such excipients as lactose or milk sugars as well as high molecular weight polyethylene glycols and the like.

[0112] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate. propylene glycol, 1,3-butylene glycol, oils (in particular, from cotton seeds, peanuts, corn, germ, oil, castor and sesame), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting, emulsifying and suspending, sweetening, flavoring, coloring, flavoring and preserving agents.

[0113] Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0114] The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be guaranteed by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0115] It is understood that the dosage regimen will be determined by the attending physician, taking into account various factors that modify the activity of the subject compounds of the invention (e.g., ActRIIa and ActRIIb polypeptides). Various factors include, but are not limited to, the patient's red blood cell count, hemoglobin level or other diagnostic assessments, the desired target red blood cell count, patient's age, sex and diet, the severity of any disease that may contribute to a reduction in red blood cell count, time applications and other clinical factors. The addition of other known growth factors to the final composition may also affect dosage. Progress can be monitored by periodic assessment of red blood cell and hemoglobin levels as well as reticulocyte levels and other indicators of the hematopoietic process.

[0116] Experiments on primates and humans have shown that the effects of ActRIIa-Fc interaction on red blood cell levels are detectable when the compound is dosed at intervals and in sufficient amounts to achieve serum concentrations of about 100 ng / ml or higher for a period of at least about 20 to 30 days. Dosages up to serum levels of 200 ng / ml, 500 ng / ml, 1000 ng / ml or higher may also be used for a period of at least 20 to 30 days. Bone effects can be observed at serum levels of about 200 ng / ml, with significant effects beginning to occur from about 1000 ng / ml or above for a period of at least about 20 to 30 days. In this way, if it is desired to achieve an effect on red blood cells with little effect on the bone, a dosing regimen can be designed to provide a serum concentration of between about 100 and 1000 ng / ml for a period of about 20 to 30 days. In humans, serum levels of 200 ng / ml can be achieved with a single dose of 0.1 mg / kg or more, and serum levels of 1000 ng / ml can be achieved with a single dose of 0.3 mg / kg or more. The observed serum half-life of the molecule is between about 20 and 30 days, substantially longer than most Fc fusion proteins, and therefore a persistent, effective serum level can be achieved, for example, by dosing about 0.05 to 0.5 mg / kg on a weekly or bi-weekly basis, or higher doses may be used with longer dosing intervals. For example, doses of the order of 0.1 to 1 mg / kg may be used on a monthly or bi-monthly basis.

[0117] We disclose gene therapy for in vivo production of ActRII polypeptides. Such therapy would achieve its therapeutic effect by introducing ActRIIa or ActRIIb polynucleotide sequences into cells or tissues having the disorder as detailed above. Delivery of ActRII polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or colloidal dispersion system. For therapeutic delivery of ActRII polynucleotide sequences, the use of targeted liposomes is preferred.

[0118] Various viral vectors that can be used for gene therapy as described herein include adenoviruses, herpes virus, bovine pox virus or RNA virus such as retrovirus. The retroviral vector may be a derivative of a murine or bird retrovirus. Examples of retroviral vectors into which a single, foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), murine Harvey sarcoma virus (HaMuSV), murine breast cancer virus (MuMTV) and sarcoma virus Rousa (RSV). A number of additional retroviral vectors can contain multiple genes. All of these vectors can carry or incorporate the selectable marker gene so that cells can be identified and generated after such transduction. Retroviral vectors can be made specific to the target by attaching, for example, a sugar, glycolipid or protein. Preferred targeting is achieved using an antibody. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retrovirus genome or attached to a viral envelope to allow target-specific delivery of a retroviral vector containing an ActRII polynucleotide.

[0119] Alternatively, tissue culture cells can be directly transfected with plasmids encoding structural gag, pol and env retrovirus genes by conventional calcium phosphate transfection. These cells are then transfected with a plasmid vector containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

[0120] Another targeted delivery system for ActRII polynucleotides is the colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, spheres, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Preferably, the colloidal system is liposome. Liposomes are artificial membrane vesicles that are useful as in vitro and in vivo delivery vehicles. RNA, DNA and intact virions can be encapsulated in an aqueous interior and delivered to cells in a biologically active form (see, e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficiently transferring genes using a liposome carrier are known in the art, see, e.g., Mannino, et al., Biotechniques, 6: 682, 1988. The liposome composition is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0121] Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. It is also possible to target liposomes, based on, for example, organ specificity, cell specificity and organelle specificity, and is known in the art.

EXAMPLES [0122] The invention described in general will become better understood by reference to the following examples, which are provided for purposes of illustration only of some embodiments of the invention, and are not intended to limit the scope of the invention.

Example 1: ActRIIa-Fc fusion proteins [0123] Applicants constructed a soluble ActRIIa fusion protein that has the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with a minimal linker between them. The constructs are designated ActRIIa-hFc and ActRIIa-mFc, respectively.

[0124] ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO: 7):

ILC RSETQEC LFFN AN WE KDRTN QTGVEPCYGDKDKRRH CF ATW K NISGSI El V KQG CW LD DIN CYD RTDC VEKKDS P Π VY FCCCEGEGMC N EKFS YF F t

NWYV DG YEYHN A KTKP R EEOYN STY RVV SVL TV1. HODW vol. NC KFY KCKYSN KALP

V E1ĘKJISK AKGOFREFOYYlfLP P_SRT. £ MIS NDYStECLY KG FY PSD1AY EWESNGOP

ΕΝΝΥΚ'Π PPV1-DSDGSFFLVSKLTVDKSRWOOGNVFSCSVMHEA1.HNHYTQKSLSL

SPGK [0125] ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Three different leader sequences were considered:

(i) Bee honey melitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ

ID NO: 8) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 9) (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 10).

[0126] The selected form uses the TPA leader and has the following untreated amino acid sequence:

M DAM KRG LCC VLLLCG A VF VSP GA AILG RSETQEC LFF N AN W EK D RTNQTG V EPC YG DK.DKR ​​R HCFAT W KNISGS [ΕIVKQG CW LDDINC YD RTD CV EKK DSR Ε V YFCCCEG N MC NE KFS Y FPE ME VTQPTSN KPPTG GGTH1 CP PCP APE LLGG PS VF LFPPKPK DT LMIS RTP E VTC VVVDVSH EDP Ε V KFN W Υ V DG VĘ V HN AKTK P REEQ YN5T YRVV 5 VLT VL H QDW LNGK E YKCK VSN K LP VP VP YTLPP SR Ε EMTKN Q VS LTC LVKG F YRSD1A VE WESNG QPENN Υ KTTP PV LDSDGS FFI, Y SKLTVD K RR WQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13) [0127] This polypeptide is encoded by the following nucleic acid sequence:

<img file="PL2124999T3_D0001.tif" />

<img file="PL2124999T3_D0002.tif" />

[0128] Both ActRIIa-hFc and ActRIIa-mFc were extremely susceptible to recombinant expression. As shown in Figure 1, the protein was purified as a single, well-defined protein peak. N-terminal sequencing revealed a single ILGRSTQE sequence (SEQ ID NO: 11). Purification can be accomplished by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be completed by virus filtration and buffer exchange. The ActRIIa-hFc protein was purified to&gt; 98% purity as determined by size exclusion chromatography and&gt; 95% as determined by SDS PAGE.

[0129] ActRIIa-hFc and ActRIIa-mFc showed high affinity for ligands, especially activin A. GDF-11 or activin A ("ActA") were immobilized on a Biacore CM5 chip using a standard amine coupling procedure. ActRIIa-hFc and ActRIIa-mFc proteins were applied to the system, and binding was measured. ActRIIa-hFc binds to activin with a 5 x 10 dissociation constant (KD)^-12and the protein bound to GDF11 with KD 9.96 x 10^-⁹. See figure 2. ActRIIa-mFc behaved similarly.

[0130] ActRIIa-hFc was very stable in pharmacokinetic studies. Rats were dosed with 1 mg / kg, 3 mg / kg or 10 mg / kg ActRIIa-hFc protein and plasma protein levels were measured after 24, 48, 72, 144 and 168 hours. In a separate study, rats were dosed with 1 mg / kg, 10 mg / kg or 30 mg / kg. In rats, ActRIIa-hFc had an 11-14 day serum half-life, and circulating drug levels were quite high after two weeks (11 μg / ml, 110 μg / ml or 304 μg / ml with initial administration of 1 mg / kg , 10 mg / kg or 30 mg / kg, respectively). In cynomolgus monkeys, the plasma half-life was significantly longer than 14 days and the drug levels in the circulatory system were 25 μg / ml, 304 μg / ml or 1440 μg / ml at the initial dosing of 1 mg / kg, 10 mg / kg or 30 mg / kg, respectively.

Example 2: ActRIIa-hFc increases red blood cell levels in non-human primates. [0131] Four groups were used in the study, five male and five female cynomolgus monkeys each, of which three individuals of each sex per group were destined to be sacrificed on day 29, and two individuals per sex per group were destined for killing on Day 57. Each animal was administered vehicle (Group I) or ActRIIa-Fc at doses of 1, 10 or 30 mg / kg (Groups 2, 3 and 4, respectively) by intravenous (IV) injection on days 1, 8, 15 and 22. Dose volume maintained at 3 ml / kg. Different measurements of red blood cell levels were carried out two days before the first administration and on days 15, 29 and 57 (the other two animals) after the first administration.

[0132] ActRIIa-hFc causes a statistically significant increase in the mean of red blood cell parameters (red blood cell count [RBC], hemoglobin [HGB] and hematocrit [HCT]) in males and females at all dose levels and time points throughout the study period , accompanied by an increase in the absolute and relative reticulocyte count (ARTC; RTC). See Figures 3-6.

[0133] Statistical significance was calculated for each treatment group, relative to the mean for the treatment group at the start of the study.

[0134] It is worth noting that the increase in red blood cell counts and hemoglobin levels is more or less equivalent in magnitude of the effects of the effects reported for erythropoietin. These symptoms occur more quickly with ActRIIa-Fc than with erythropoietin.

[0135] Similar results were seen in rats and mice.

Example 3: ActRIIa-hFc increases red blood cell levels in human patients [0136] The ActRIIa-hFc fusion protein described in Example 1 was administered to human patients in a randomized, double-blind, placebo-controlled study that was conducted to assess, mainly, safety protein in healthy postmenopausal women. Forty-eight patients were randomized into cohorts of 6 to receive a single dose of ActRIIa-hFc or placebo (5 active: 1 placebo). Dose levels ranged from 0.01 to 3.0 mg / kg intravenously (IV) and 0.03 to 0.1 mg / kg subcutaneously (SC). All patients were followed for 120 days. In addition to pharmacokinetic (PK) analyzes, the biological activity of ActRIIa-hFc was also assessed by measuring biochemical markers of bone formation and resorption, and FSH levels.

[0137] To observe potential changes, hemoglobin levels and RBC counts in all patients were analyzed in detail for the duration of the study and compared to baseline. Platelet counts were compared for the same time as the control. No clinically significant changes were observed over time relative to baseline values ​​for platelet counts.

[0138] PK Analysis of ActRIIa-hFc presented a linear dose-profile, and mean half-life of approximately 25-32 days. Area under the curve (AUC) for ActRIIa-hFc was linear with the dose and absorption after SC dosing was essentially complete (see Figures 7 and 8). These data indicate that SC is a desirable dosing approach as it provides equivalent bioavailability and serum half-life, while avoiding a sharp increase in serum drug levels associated with the first few days of IV dosing (see Figure 8). ActRIIc-hFc caused a rapid, dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BAP), which is a marker for anabolic bone growth, and a dose-dependent decrease in levels of C-terminal collagen telopeptide, type 1 and tartrate-resistant acid phosphatase 5b which are bone resorption markers. Other markers such as P1NP showed inconclusive results. BAP levels showed an almost full saturation effect at the highest dose of drug, indicating that half of the maximal effect on this anabolic biomarker bone could be achieved at a dose of 0.3 mg / kg, with an increase to 3 mg / kg. Calculated as the ratio of pharmacodynamic effects to drug AUC, the EC50 was 51.465 (day * ng / ml). See Figure

9. These bone biomarker changes were maintained for approximately 120 days at the highest dose levels tested. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin.

[0139] Overall, a very small, drug-related reduction in hemoglobin levels was observed during the first week of the study, probably related to blood sampling for the study, in the 0.01 and 0.03 mg / kg groups, whether collected IV or SC . Hemoglobin levels for 0.1 mg / kg SC and IV were stable or showed a slight increase until day 8-15. At the 0.3 mg / kg IV dose level, a clear increase in HGB level was observed, visible as early as on day 2 and often reaching a peak on day 15-29 that was not seen in patients taking placebo. For this point in the study, this change did not reach statistical significance.

[0140] Overall, ActRIIa-hFc showed a dose-dependent effect on red blood cell count and reticulocyte count. For a summary of hematological changes, see Figures 10-13.

Example 4: Alternative ActRIIa-Fc proteins [0141] A number of ActRIIa variants that can be used in accordance with the methods described herein are presented in International Patent Application, published as WO2006 / 012627 (see e.g. pp. 55-58). The alternative construct may have a deletion of the c-terminal tail (terminal 15 amino acids of the extracellular domain of ActRIIa. The sequence of such a construct is shown below (part Fc underlined) (SEQ

ID NO: 12):

and LG RS ETQEC LFFN AN W EKDRTNQTGVEPC YGDKDKR RIICF ATWK N1SGSIE1VKQG C WLD D INC YD RTDC VE KKDS FE VYFCCC EGNM CN EKF S YFPEMTG GGTHTC F PC? A PE.UGGPSYFLFFPKPW1'LMISRTPEYTCVVVDVSHEE1PEVKFNWYVDGVEVHNAK

TKP REEQ YN s T YR V VSVLT VJ-H QD WLNOKE YKCK VSN KA LP VP [EKTIS KAKGOP BE

FOVYTLPP5RFEMTKNOVSLTCLVftGFY? SD? AVEWĘSNGC) PENNYKTTPPVLDSPG SFFL YS K. 1. TV DK a R WQpGNV FSC a V ΜΗ K AI. HN H VTQ KS LS IS Pf SK

Example 5: ActRIIb-Fc fusion proteins [0142] Applicants constructed a soluble ActRIIb fusion protein that has the extracellular domain of human ActRIIb fused to a human Fc domain. The co-crystal structure of activin and extracellular ActRIIb did not show any terminal function (C-terminus) of 15 amino acids (called "tail" in the description) of the extracellular domain in ligand binding. This sequence could not be resolved by the crystal structure, suggesting that these residues are present in a flexible loop that was not uniformly packed in the crystal. Thompson et al. EMBO J. 2003 Apr 1; ​​22 (7): 155525 66. This sequence is also poorly preserved between ActRIIb and ActRIIa. Accordingly, these residues were omitted from the primary or secondary ActRIIb-Fc fusion construct. In addition, position 64 in the background form is occupied by alanine, which is generally considered the "wild type" form, although the A64R allele occurs naturally. In this way, the secondary ActRIIb-Fc fusion has the sequence (Fc part underlined) (SEQ ID NO: 20):

EG RGE A ETR ECf Y YN AN W ELE R TNQSG LE R CEGEQDKRLHC YASW ANS5GTIEL νΚ KGCW LD D FN CY DR QEC V AT E ENPQ VY l-CCCEGN FCN E RFT HL Ρ EA OGGTHTCPPCP AEELi.OgPS VFLPPPM £ HE DFEVKFN W YV DG VE VHNA KTKPREEOYN S.TYR VVS VLTV LHO LNGKEY KC K VSNKA LP VP fę K.TI S K. ΑΚΏΟ P

R £ PQVYTLPPSRFEMTKiMOVSLTCLVKGFYPSniAVeWESNOOPEWYXTTPPVLDS

DG 5 FFL Y 5 K LT VDK SR WOOGN YFS CS YM J and EA LH NH YTOK SLSLSFGK [0143] Surprisingly, it was found that the tail at the C-terminal strengthened the binding of activin and GDF-11, thus the preferred version of ActRIIb-Fc has sequence (part Fc underlined) (SEQ ID NO: 21):

<img file="PL2124999T3_D0003.tif" />

A number of ActRIIb variants that can be used according to the methods described herein have been presented in the international patent application published as WO2006 / 012627 (see e.g. pp. 59-60).

Example 6: ActRIIb-hFc stimulates erythropoiesis in non-human primates [0144] ActRIIb-hFc (IgG 1) was administered once weekly for 1 month to male and female cynomolgus monkeys by subcutaneous injection. Forty-eight cynomolgus monkeys (24 / sex) were assigned to one of four treatment groups (6 animals / sex / group) and were given subcutaneous injections or vehicles or ActRIIb-hFc at 3, 10 or 30 mg / kg once weekly for 4 weeks (total, 5 doses). The parameters assessed included general clinical pathology (hematology, clinical chemistry, coagulation and urinalysis). ActRIIbhFc caused a statistically significant increase in mean reticulocyte absolute values ​​up to day 15 in treated animals. By day 36, ActRIIb-hFc caused major haematological changes, including an increase in the reticulocyte absolute value and red blood cell volume distribution range and a decrease in the mean hemoglobin concentration in erythrocytes. The changes affected all treated groups and both sexes. These effects are consistent with the positive effect of ActRIIb-hFc on the release of immature reticulocytes from the bone marrow. This effect was reversed after drug elution in treated animals (up to day 56 of the study). Accordingly, it was concluded that ActRIIb-hFc stimulates erythropoiesis.

[0145] While specific embodiments of the subject matter have been discussed, the above specification is illustrative rather than limiting. Many variants will be apparent to those skilled in the art upon reading this specification and the following patent claims. The full scope of the invention should be determined with reference to the claims and specifications.