AU2013221910A1 - Activin-ActRII antagonists and uses for increasing red blood cell levels - Google Patents

Abstract

In certain aspects, the present invention provides compositions and methods for increasing red blood cell and/or hemoglobin levels in vertebrates, including rodents and prirnates, and particularly in humans

Description

ACTIVIN-ACTRII ANTAGONISTS AND USES FOR INCREASING RED BLOOD CELL LEVELS RELATED APPLICATIONS The present application is a divisional application of Australian Application 5 No. 2007334333, which is incorporated in its entirety herein by reference. This application claims the benefit of U.S. Provisional Patent Application No. 60/875,682, filed December 18, 2006, which application is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION 0 The mature red blood cell, or erythrocyte, is responsible for oxygen transport in the circulatory systems of vertebrates. Red blood cells carry high concentrations of hemoglobin, a protein that binds oxygen in the lungs at relatively high partial pressure of oxygen (pO2) and delivers oxygen to areas of the body with a relatively low pO2. Mature red blood cells are produced from pluripotent hematopoietic stem cells in a 5 process termed erythropoiesis. In post-natal individuals, erythropoiesis occurs primarily in the bone marrow and in the red pulp of the spleen. The coordinated action of various signaling pathways control the balance of cell proliferation, differentiation, survival and death. Under normal conditions, red blood cells are produced at a rate that maintains a constant red cell mass in the body, and production may increase or decrease in response to various stimuli, including 20 increased or decreased oxygen tension or tissue demand. The process of erythropoiesis begins with the formation of lineage committed precursor cells and proceeds through a series of distinct precursor cell types. The final stages of erythropoiesis occur as reticulocytes are released into the bloodstream and lose their mitochondria and ribosomes while assuming the morphology of mature red blood cell. An elevated level of reticulocytes, or an elevated 25 reticulocyte:erythrocyte ratio, in the blood is indicative of increased red blood cell production rates. Erythropoietin (Epo) is widely recognized as the most significant positive regulator of erythropoiesis in post-natal vertebrates. Epo regulates the compensatory erythropoietic response to reduced tissue oxygen tension (hypoxia) and low red blood cell levels or low 30 hemoglobin levels. In humans, elevated Epo levels promote red blood cell formation by stimulating the generation of erythroid progenitors in the bone marrow and spleen, In the mouse, Epo enhances erythropoicsis pri marily in the spleen. Various forns of reconibinant Epo are used by physicians to increase red blood cell levels in a variety of clinical settings, and particularly for the treatment of anemia. Anemia is 5 a broadly-defined condition characterized by lower than normal levels of hemoglobin or red blood cells in the blood, In some instances" anemia is caused by a primary disorder in the production or survival of red blood cells. More commonly, anemia is secondary to diseases of other systems (Weatheral I & Provan (2000) Lancet 355, 11691175). Anemia may result from a reduced rate of production or increased rate of destruction of red blood cells or by loss 10 of red blood cells due to bleeding Anemia may result from a variety of disorders that include, for example, chronic renal failure, myerodysplastic syndrome, rheumatoid arthritis and bone marrow transplantation. Treatment with Epo typically causes a rse in henioglobins by about 1-3 g/dl in healthy humsns over a period of weeks. When administered to anemic individuals, this 15 treatment regimen otlen provides substantial increases in, hemoglobin and red blood cell levels and leads to improvements in quality of life and prolonged survival, Epo is not uniformly effective. and rimany individuals are refractory to even high doses (Horl et at (2000) Nephrol Dial Transplant 15, 43-50). Over 50% of patients with cancer have an inadequate response to Epo, approximately I0% with end-stage renal disease are 20 hyporesponsive (Glaspy et al. (1997} J Clin Oncol 15, 1218-1234; Denetri et al. (1998) J Clin Oncol 16, 3412-3425), and less than 10% with mylodysplastic syndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67), Several factors, including inflammation,iron and vitamn in deficiency, inadequate dialysis, aluminum toxicity, and hyperparathyroidism may predict a poor therapeutic response tihe molecular mechanisms of 25 resistance to Epo are as yet unclear Thus, it is an object of the present disclosure to provide alternative compositions and methods for increasing red blood cell levels in patients, SUMMARY OF THE INVENTION In part, the disclosure demonstrates that activin antagonists, as well as ActRIla and ActRllb antagonists, can be used to increase red blood cell and hemoglobin levels. In particular, the disclosure demonstrates that a soluble form of ActRila acts as an inhibitor of 5 activin and, when administered in vivo, increases red blood cell levels in the blood A milder effect was observed with a soluble frm of Atllb, which binds Activin A with lesser affinitythan soluble ActRlla. While soluble ActRila and ActRb may affect red blood cell levels through a mechanism other than active ant agonism, the disclosure nonetheless demonstrates that desirable therapeutic agents may be selected on the basis of activin 10 antagonism or ActRil antagonism or both Such agents are referred to collectively as acivin ActRIIantagonists. Therefore in certain embodiments, the disclosure prvdes methods for using acti-viActRH antagonistsincluding, for example, activin-binding ActR11a polypeptides, activin-bindi ng ActRUb polypeptides, anti -activin antibodies, anti-ActR ia antibodies, anti Atllb antibodies. activin-, ActRUb-. or Actklatargeted small molecules 15 and aptaners and nucleic acids that decrease expression of action, AciRilb, or ActRIla, to increase red blood cell and hemoglobin levels in patients and to treat disorders associated with low red blood cell or hemoglobin levels in patients in need thereof As described in U.S. Patent Application Serial No. 11/603,485, incorporated by reference herein, activir-ActRa antagonists can be used to promote bone growth and increase bone density As described 20 herein the effects of such antagonists on red blood cell levels are more rapid and occur at lower doses than the effects of such antagonists on bone. Thus, in certain embodiments the disclosure provides methods for using an activin-Act RIla antagonist to increase red blood cell or hemoglobin levels without causing a significant increase in bone density, For example, a method may cause less than 3%, 5%, 10% or 15% increase in bone density, '[his selective 25 effet may be achieved by using, for example, lower doses of activin-ActR a antagonist, less frequent doses, or by using an activinActRila antagonist with a shorter serum hallife at doses and frequencies calculated to provide a lower serum concentration. In certain aspects, the disclosure provides polypeptides comprising a soluble, activin binding ActRII polypeptide that binds to activin. The activin binding polypeptide may be an 30 ActR la polypeptide or an ActRllb polypeptide, ActRIl polypeptides may be formulated as a pharmaceutical preparation comprising the activin-hinding ActRIl polypeptide and a -3, pharmaceutically acceptable carrier. The activin-binding ActRU pol ypeptide rnay bind to actim with a K less than I nicronolar or less than 100, 10 or I nanomolar; Optionally, the adpolypeptide selectively binds aetivn versus GDFI I and/or ODF8 and optionally with a Ko that is at least 10-fold, 204old or 504bId lower with respect to activin 5 than with respect to 3DFI I and/or GDF8 While not wishing to be bound to a particular mechanism of action, it is expected that this degree of sdeetivity br activin inhibition over GIM." ODES inhibition accounts for effects on bone or erythropoiesis without a consistently measurable effect on muscle, In many embodimentsan ActR.Il polypeptide will be selected tor causing less than 15%, less than 10% or less than 5% increase in muscle at doses that 10 achieve desirable effects on red blood cell levels, The composition may be at least 95% pure, with respect to other polypeptide components as assessed by size exclusion chromatography, and optionally, the composition is at least 98% pure, An activin-binding ActRIla polypeptide for use in such a preparation may be any of those disclosed herein, such as a polypeptide having an amino acid sequence selected fron SEQ ID N0s: 2,3, 7 or 12, or having anmino 15 acid sequence thats at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 12 or 13. An activin-bindingActRlHa polypeptide may include fictional fragment of a natural ActRnla polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected front SEQ ID NOs: 1-3 or a sequence of SEQ ID NO: 2. lacking the Cterninal 10 to 15 amino acids (the "tail" An 20 active binding ActRllb polypeptide for use in such a preparation may be any of those disclosed herein, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 16 17, 20, or 21 or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 16, 17, 20, or 21. An activin-binding ActRiIb polypeptide may inchide a functional fragment of a 25 natural ActRIlb polypeptide, such as one comprising at least 10, 20 or 30 amino acids of SEQ 1D NOs: 15-17 or a sequence lacking the Cterminal 10 to IS amino acids (the "tail such as SEQ I) NO: 17. A soluble active inbinding ActRilI polypeptide may include one or more alerations in the amino acid sequence (eg, in the ligand-binding domain) reladiveto a naturally occurring 30 ActRil polypeptide, Examples of altered ActRIla and ActRllb polypeptides are provided in WO 2006/012627 pp. 59-60 and pp 55 -58, respectively, which is incorporated by reference herein; The alteration in the amino acid sequence may, for example, alter glycosylation of A4 the polypeptide when produced in a mammalian, insect or other eukaryotic cell or alter proteolytic leavage of the polypeptide relative to the naturally occurring ActR11 polypeptide. An activin-binding ActRi polypeptide may be a fusion protin that has, as one domain, an ActRII polypeptide {e .g.,a ligand-binding portion of an ActRla or ActRlb) and 5 one or more additional domains that provide a desirable property, such as improved pharmnacokinetics;, easier purification targeting to particular tissues. etc. For example a domain of a fusion protein may enhance one or more of in vivo stability in vivo half ife, uptakeadm inistratin tissue localization or distribution, fon-nation of protein complexes multimerization of the fusion protein/and/or puriticatiori An activin-biading ActR i fusion 10 protein may include an mmunoglobulin Fe domain (wild-type or Iutant) or a serum albumin or other polypeptide portion that provides desirable properties such as improved pharmacokinetics, improved solubility or improved stability In a preferred embodiment, an ActRIlEFc fusion comprises a relatively unstructured linker positioned between the Fc domain and the extracelular ActRll domain. This unstructured linker may correspond to the 15 roughly 15 amino acid unstructured region at the Ct terminal end of the extracellular domain ofActRIl (the or it may be an artificial sequence of1, 2, 3, 4 or 5 amino acids or a length of between 5 and 15, 20, 30, 50 or more amino acids that are relatively free of secondary structure, or a mixture of both. A tinker may be rich in glycine and proline residues and may, for example, contain a single sequence of threonine/serine and glycines or 20 repeating sequences of threonine/serine and glycines (e~g, Tg0n or S 4 singlets or repeats), A fusion protein may include a purification subsequencesuch as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. Optionally, a soluble ActRII polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid a PEylated amino acid, a farnesylated amino acid, an acetylated annno acid, a biotinylated. 25 amino acid, an amino acid conjugated to a lipid moiety, and an amino acid coniugated to an organic derivatizing agent, A pharrmaceuticalpreparation may also include one or more addition compounds such as a compound that is used to treat a bone disorder Preferably, a pharmaceutical preparation is substantially pyro generalit is preferable that an ActRil protein be expressed in a mammalan cel line that mediates suitably natural 30 glycosylation of the ActRlU protein so as to diminish the likeliood of an unfavorable immune response in a patient Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression systems will be useful.

As described herein, Actluia proteins designated ActRIla-Fc (a form with a minimal linker between the ActRfla portion and the Fe portion have desirable properties. including ) selective binding to activin versus GDF8 and/or GDFI high affinity ligand binding and serum half life greater than two weeks in animal models. In certain embodiments the 5 invention provides ActRIb-Fc polypeptides and pharmaceutical preparations comprising such polypeptides and a pharmaceuticals acceptable excipient, In certain aspects, the disclosure provides nucleic acids encoding a soluble activin binding ActR ii polypeptide, such as an AetRila or ActPlb polypeptide An isolated polyn ucleotide may comprise a coding sequence tor a soluble, activn-binding ActR1l 10 polypeptide, such as described above. For example, an isolated nuclei acid may include a sequence coding or an extracellular domain (eig. ligand-binding domain) of an ActRli and a sequence that would code for part or all of the trainsmiembrane domain and/or the cytoplasmic domain of an ActR 1t but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the 15 transmembrane domain or cytoplasmic domain For examplean isolated polynucleotide may comprise a Ifull-kngh AetRl a polynudeotide sequence such as SQ ID NO: 4 or 5 or a full length ActRllb polynucleotide sequence such as SEQ D NO: I8 or a partially truncated version of ActRIla or ActRIb, said isolated polynucleotide further comprising a transcription termination codon at least six hundred nucleotides before the 3 t terminus or otherwise 20 positioned such that translation of the poiynucleotide gives rise to an extracellular domain. optionally fused to a truncated portion of a fulliength ActRIL A preferred nucleic acid sequence for Act Rla is SEQ ID NO: 14, Nucleic acids disclosed herein may be operably linked to a promoter for expression, and the disclosure provides celIs transformed with such recombinant polynucleotides, Preferably the cell is a mammalian cell such as a CHO cell 25 In certain aspects, the disclosure provides methods for making a soluble, activin binding ActR i poiypeptide Such a method may include expressing any of the nudeic acids (eg, SEQ D NO: 4, 5 14, 18, or 19) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) edl Such a method may comprise a) culturing a cell under conditions suitable for expression of the soluble ActRl polypeptide, wherein said cell is transfbrnmcd 30 with a soluble AetRil expression construct; and b) recovering the soluble ActRl polypeptide so expressed. Soluble ActRil polypeptides may be recovered as crude, partially purified or highly purified fractions, Purification ma'y be achieved by a series of purification steps, including, Forexample 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 hrmoap and 5 cation exchange chromatography. In certain aspects, activinAetR1lantagonist disclosed herein, such as a soluble, activin-binding ActRUa polypeptide or soluble activirn-inding ActRlb polypeptide, may be used in a method for promoting red blood cell production or increasing red blood cell levels in a subject. In certain embodiments, the disclosure provides methods for treating a dir 10 associated with low red blood cel counts or low hemoglobinlevels (eg, an anemia), or to promote red blood cell production, in patients in need thereof A method may comprise administering to a subject in need thereof an effective amount of activinActRII antagonist. In certain aspects, the disclosure provides uses of activin~ActRII antagonists for making a medicament for the treatment of a disorder or condition as described herein; 15 In certain aspects, the disclosure provides a method for identifying an agent that stimulates production of red blood cells. The method comprises: a) identifying a test agent that binds to activin or a ligand-binding domain of an ActRU polypeptide; and b) evaluating the effect of the agent on the levels of red blood cells, hemoglobin, and/or red blood cell precursor levels (e.g., reticulocyte levels), 20 BRIEF DESCRIPTION OF'THE DRAWINGS Figure 1 shows the purification of ActRila-hFc expressed in CHO cells, The protein purifes a single welldefied peak as visualized by sizing colrn (left panel)and Coomassie stained SDS -P AGE (right panel) (left lane: molecular weight standards; right 25 lane: ActRIla-hEFc) igure 2 shows the binding of ActRia-hFc to activin and CDF-1 as measured by ffiaCoreM assay. Figure 3 shows the effects of ActRhahFc on red blood cell counts in female non human primates. Female cynomolgus monkeys (fIour groups of fAve monkeys each) were 30 treated with placebo or 1 mg/kg; 10 .mgkg or 30 mg/kg of AcR I la-hFc on day 0, day 7, day l4 and day 2l. Figure 3A shows red blood cell (RBC) counts Figure 3B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group, At day 57, ) two monkeys remained in each group, Figure 4 shows the effects of ActRIla-hFc on red blood cell counts in male non 5 human primates. Male cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or I mg/kg, 10 mg/kg or 30 mg/kg of ActRlla-hFc on day 0, day 7, day 14 and day 21 Figure 4A shows red blood cell (RBC) counts. Figure 4B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group. 10 Figure 5 shows the effects of ActRIIa-hFc on reticulocyte counts in female non human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or I mg/kg, 10 mg/kg or 30 mg/kg of ActR la-hFc on day 0, day 7, day 14 and day 21~ Figure 5 A shows absolute reticulocyte counts, Figure 5B shows the percentage of reticulocytes relative to RIBCs. Statistical significance is relative to baseline for each group. 15 At day 57, two monkeys remained in each group. Figure 6 shows the effets of ActRIia-hFc on reticulocyte counts in female non human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or I mg/kg, 10 mg/kg or 30 mg/kg of ActRlIa-hFe on day 0, day 7, day 14 and day 2 L Figure 6A shows absolute reticulocyte counts. Figure 6B shows the percentage of 20 reticulocytes relative to RBCs. Statistical significance is relative to basehme for each group. At day 57, two monkeys remained in edach group Figure 7 shows results from the human clinical trial described in Example 5, where the area-under-curve (AUC) and administered dose of ActRIla-hFe have a linear correlation. regardless of whether ActRI a-hFc was administered intravenously (IV) or subcutaneously 25 (SC), Figure 8 shows a comparison of serum levels of ActRlla-hFc in patients administered IV or SC. Figure 9 shows bone alkaline phosphatase (BAP) levels in response to different dose levels of ActRlla-hFc. BAP is a marker for anabolic bone growth. 30 Figure 10 depicts the median change from baseline of hematocnt levels from the human clinical trial described in Example 5, AczRla-hFc was administered intravenously (IV) at the indicated dosage.

Figure l I depicts the median change from baseline of hemoglobin levels from the human clinical tial described in ExampleY5ActRllahFc was administered ininwenously (IV) at the indicated dosage Figure 12 depicts the median change from baseline of RBC red blood cell) count 5 from the human clinical trial described in Eamiple 5 ActRia-hF.e was administered intravenously (IV) at the indicated dosage. Figure 13 depicts the median change from baseline of reticulocyte count from the human clinical triAl deseibed in Example 5. ActRlahne was administered intravenously (IV) at the indicated dosage, 10 DETAILED DESCRIPTION OFTHE INV ENTION I. Overview The transferring growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins 15 are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important funetons during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenests, chondrogenesis, card genesis, hermatopoiesisneurogenesis, and epithelial cell differentiation. The family is 20 divided into two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10 branches, whose members have di verse. often complementary effects. By manipulating the activity of a member of the TGF-eta family, it is often possible to cause significant physiologicalchanges in an organism For example, the Piedmontese and Begian Blue cattle breeds carry a los-of4unction mutation in the ODF8 (also called myostatin) gene that causes 25 a marked increase in muscle nass. Grobet et al., Nat Genet, 1997, (1), 1 4. Furthermore, in humans, inactive alleges of GDES are associated with increased muscle mass and, reportedly, exceptional strength Schuelke et al N Eng Med 2004, 350:2682.

Activins are dimerc polypeptide growth factors that belong to the To1'GFeta superimil]y. There are three principal activin forms (A, B. and AB) that are 30 homo/heterodimers of two closely related P subunits (PAPA, PO@E and Ag43 respectively) The human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing Pr or fiare, also knowns in the TOF-beta superfannily, activns are unique and multifrnctional factors that can stimulate hormone production in ovarian and placental cells) support neuronal cell surval influence 5 cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in aaphibian embryos (DePaolo t aL, 1991, Proc Soc Ep Biol Med. 198:00-512: Dyson et at, 1997Curr Biot 781-84; Woodruff, 1998, Biochem Pharnacot 55953~963) Noreoer, erythroid differentiation factor (EDF) isolated from the stinulated human monocytic icukemic cells was found to be identical to activin A (Murata et al.. 1988 10 PNAS, 85:2434). It has been suggested that activin A promotes erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its related heterodiimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary, activin promotes FSR secretion and synthesis, while inhibin prevents FPSH secretion and synthesis. Other proteins that may regulate activin bicactivity and/or bind to activin 15 include f1istatin (FSf) tolistatin-rel ated proteiii (FSRP) and a 2 -nacroglobulin. TGPi3 signals are mediated by heteromeric complexes of type I and type I1 shrine/ threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massagu 0 200, Nat. Rev Mot Cell Biol, 1:169-178 These type I and type Ii receptors are transmemibrane proteins, composed of a ligand -binding extracellular 20 domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specifcity, Type I receptors are essential for signaling; and type II receptors are required for binding lgands and for expression of type I receptors. Type I and 1. activin receptors form a stable complex after grand hiding, resulting in phosphorylation of type I receptors by type ll receptors. 25 Two related type ii receptors (ActR l) ActRIa id AitRb have been identified as the type II receptors for activins (Mathews and Vale, 1991, Cel 697-982: Attisanoet at. 1992, Cell 68 9-108). Besides activist, ActR1la and ActRlb can biochemicaly interact with several other TGFI family proteins, including BMP7, Nodal, 0DF, and GDFf I (Yamashita ct al, 1995, J Cell RioL 130:21 226; Lee and McPherton, 2001.Proc Nati, 30 Acad. Si. 98:9306-9311; Yeo and Whitman, 2001, M Cell 7 949-957; Oh et al, 2002, Genes Dev, 16 :274954). ALK4 is the primary type Ireceptor for activins, particularly for actinAand ALK-7 may serve as a receptor for activi us as well, particularly for activin B As demonstrated herein, a soluble ActRa polypeptide (sActRila), which shows substantial preference in binding to activin A as opposed to other TGFbeta firnily members, 5 such as GDE& or CDFI 1, is effective to increase red blood cell levels in vivo. While not wishing to be bound to any particular necharism, it is expected that the effect of sActRla is caused primarily by an activin antagonist effect; given the very strong activin binding (picomolar dissociation constant) exhibited by the particuLar sActRila construct used in these studies Regardless of rnechanisnt it is apparent front this discosure that ActRIa-activin 10 antagonists increase red blood cell levels inrodents, monkeys and humans. It should be noted that hematopoiesis is a complex process, regulated by a variety of factors, including erythropoietin, GCSF and iron homeostasis. The terms "increase red blood cell levels" and "promote red blood cell formation" refer to clinically observable metries, such as hematocrit, red blood cell counts and bemoglobin measurements, and are intended to be neutral as to the 15 mechanism by which such changes occur. As also demonstrated herein, a soluble ActRl lh polypeptide (sActR.lib) is effective to increase reticulocyte levels in vivo, an effect vhich over a longer time period is expected to cause increased hemotocrit levels. The data reported herein with respect to nonhuniman primates are reproducible in 20 mice, rats and humans as well, and therefore, this disclosure provides methods for using ActRI polypeptides and other activin-ActRil antagonists to promote red blood cell production and increase red blood cell levels in mammals ranging from rodents to hunus. Activin-ActRlI antagonists include, for example, activinbinding soluble ActRita polypeptides, activin-binding soluble ActRub polypeptides, antibodies that bind to activin 25 (particularly the activin A or B subunits, also referred to as 3A or pB) and disrupt ActRita and/or Act Ilb binding, antibodies that bind to ActRfla and disrupt activin binding, antibodies that bind to ActRilb and disrupt activin binding, nonantibody proteins selected for activin, ActRilb or ActR11a binding (see eg, WO 2002/088171, WO/2006/055689; and WO/2002/032925 for examples of such proteins and methods for design and selection of 30 same), randomized peptides selected for activin, Act Rib, or ActRita binding, often affixed to an Pc domain. Two different proteins (or other moieties) with active, ActRub, or ActRa binding activity, especially activin binders that block the type I (e.g, a soluble type I activin receptor) and type 11 (e.g., a soluble type 11 activin receptor) binding sites, respectively, may ) be linked together to create a bifunctional binding molecule. Nucleic acid aptamers, small molecules and other agents that inhibit the activin-ActRIl signalng axis are included as 5 activinActRil antagonists, Various proteins have activin-A ctRi. antagonist activity., including inhibin (i.e., mhibin alpha subunit), although inhibin does not universally antagonize activin in all tissues, follistatin (e.g., follistatin-288 and. follistatin-3I5), FSRP, activin C. alpha(2)-macroglobulin, and an Ml I0SA (methionine to alanine change at position 108) mutant activin A, Generally, alernative fbrns of activin, particularly those with 10 alterations in the type 1 receptor binding domain can bind to type 11 receptors and fail to form an active ternary complex, thus acting as antagonists, Additionally, nucleic acids, such as antisense molecules, sIRNAs or ribozymes that inhibit activin A, B, C or E, or, particularly, ActRila or ActRilb expression, can be used as activinActki antagonists, The activin ActR Ii antagonist to be used may exhibit selectivity for inhibiting activin-mediated signaling 15 versus other members of the TGF-beta family, and particularly with respect to GDFS and The terms used in this specification generally have their ordinary meanings in the art, wthin the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification; to provide additional 20 guidance to the practitioner in describing the compositions and methods of the invention an how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used, "About" and 'approximately" shall general nean an acceptable degree of error for the quantity measured given the nature or precision of the measurements Typically, 25 exemplary degrees of error are within 20 percent (%) preferably within 10%,and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5 fold and more preferably within 2-fold ofa given value, Numerical quantities given herein 30 are approximate unless stated otherwise, meaning that the term "about" or approximately " can be inferred when not expressly stated The methods of the invention may include steps of comparing sequences to each other, including wildype sequence to one or more mtants (sequence variants). Such comparisons typically comprise alignments of polymer sequenceseg, using sequence alignment programs and/or algorithms that are well known in the art (for example BLAST, S FASTA and MEGAGLION, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a tnmtation contains a residue insertion or deletion, the sequence alignment will introduce a "gap" (typically represented by a dashr "A") in the polymer sequence not containing the Inserted or deed residue. "Homologous in all its grammatical frms and spelling variations refers to the 10 relationship between two proteins that possess a "common evolutionary origin," including proteins rom superfanilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nuceic acids)have sequence honology, as reflected by their sequence similarity, whether in terms or percent identity or by the presence of specific residues or motifs and conserved positions. 15 The term "sequence similarity," In all its grammatical formsrefers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin. However. in monmion usage and in the instant application, the term "honologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or 20 may not relate to a common evolutionary origin 2, ActR Polype'tides in certain aspects, the present invention relates to ActRil polypeptides As used herein, the term "ActlU" refers to the hmily of type 11 activin receptors. This family 25 includes both the aetivin receptor type la and the acivin receptor type [lb. in certain aspects, the present invention relates to ActR11a polypeptides .As used herein, the term "ActRla" refers to a family of activin receptor type [[a (ActRita) proteins tr any species and variants derived from such ActR ta proteins by mutagenesis or other modification Reference to ActRUla herein is understood to be a reference to any one of the 30 currently identified fonns, Members of the ActiRla family are general transmembrane proteins, composed of a ligand-blinding extracelluar domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase ) activity The term "ActRIya polypeptide" incdes polypep tides comprising any naturally 5 occurring polypeptide of an ActRila family member as well as any variants thereof (including mutants, fragments, ftus, and peptidomimetic forms) that retain a useful activity, See for example, WO/2006/012627, For example, ActRla polypeptides include polypeptides derived from the sequence of any known ActRUa having a sequence at least about 80% identical to the sequence of an ActR1Ha polypeptide ad optionally at least 85%, 10 90%, 95% 97%, 99% or greater identity. For example, an ActRllapolypeptide of the invention may bind to and inhibit the function of an ActRita protein and/or activin. Ar ActRla polypeptide may be selected for activity in promoting red blood cell formation in vivo Examples of ActRIla polypeptides include human ActRita precursor polypeptide (SEQ ID NO:) and soluble hnman ActRila polypeptides (e g.t SEQ ID NOs: 2, 3.7 and 12) i5 The human ActRita precursor protein sequence is as fotllows: MOAAAKIAFAVFL 1SC$SGAtILGRS ETQECLFFNANWEKDRTNQTGVEP CYGDKDKRRHCFATWXNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSP EVYFCCCEGNMCNEKFSYFPEMZVTQPTSNPVTPKPPYYNi LLYSLVPL MLIAGIVTCAFWVYRHKMAYPPJVPTQDPGPPPPSPLLGLKPLQLLE 20 VKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHEN ILQFIGAEKRGTSVDVDLWLTTAFHEKGSLSOFLKANVVSWNELCHIAE TMARGLAYLHEDIPGLKOGHKPAISHROIKSKNVLLKNNL GACIADFGL ALKFEAGKSAGDTHGQVGTPRYMAPEVLEGAINFQRDAFLRID MYAMGL VLWELAS'rTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVL 25 RDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTN IT TEDIVTVVTMVTNVDFPRK ESS L (SEQ IDNOI 1) The signal peptide is single underlined; the extraceliuar domain iS in bold and the potential Nlinked glycosylation sites are double underlined. 14- The human ActR11a soluble (extracelIular) processed polypeptide sequence is as follows: T LGRSETQ5GECFFAN WEKORTNQTGVE PGYG0DiDKRRYRCFAT WKNI:1SG STEIVKQGACVL DOINC YDRTDCVEKODSPEVYFSCCCEGNMCNEKF F5.p S EMEVTQPTSNPVTPKPP (SEQ ID N 2) The C terminall "tail" of the extracedular domain is underlined, The sequence with the "tail deleted (a Al$ sequence) is as follows: I AGSET5QECLFNANWEKDSOPTNQOGVE PGDKDKRK R55 ECA TWKN ISG S I VKQGCWT.L, DDN1 D dEKSEYCCGMNKSF I10 EM (S EQ I D N 0,3) The nucleic acid sequence encoding human ActRila precursor protein is as follows (nucleotides 164-1 705 of Genbank entry NM_001616): ATGGGAGCTGCT0GCAAACI"EGGCGT T TOGCGT CT TTG T TATCTLCA PG CT ~ ~ ~ ~ ~ ~~~~G T CGTTATATTGTGTAAATGAGT G TTT T9,-"CTT 15 TATGTAATTOGGAAAAAGAOAOAACCATOATZAGTGGTGTTGAACGG TVTT AOAAAATAAACGGCAT TGT TTrC TA G GAAG A ATA 77T 0 l7 T TAT C TAATAGTGAAAAT T(7rGGCTGGATG A 7AA5TAT0T5 " T T GC77TGAGGACAAGTAATG\AAAAOGTGTT 20 C:TTAT7T0T AGAAGAAGT CACACAGCCCAGT AAATGCAGI"TAC A~CCTAAOGCAOCCCTATT AC5ACATCGTGCTCTA TT 7CT0TG37CCA5T7T ATGT TAATTGCGGGGATTGATTTGTGATTTTGGGTTACAGGCATC ACAAGATGGCCTA7C0C7TCTGTAGTTGT TGGAACTGAAGAAGG A5000CGT0TGT 00ATTAC T5AGGGT TG5AACC0A0TGCATTTTAGA 25 GTGA CAGGAAT GTT TCGAAGCCGTT TT TT GA,5001 30 T IT T OAAGCT5AATGGTT GT CTTGAATTCA -C 11 TGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGTTG GCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGCGATACCCATGGAGAGG TTGGCTACCCGCAGCTACATGGCTCCAGAGGTATTAGAGGGTGCTATAAA CTTCCAAAGGGATGCATTTT'GAGGATACIATCTATGCCATGGATTA S~GTCTATGGGAACTGGCTTACTCGTGTACTGCTGCAGATGGACCTGTAG ATGAAACATGTTGCCATTGAGGAGGAAATTGGCCAGCATCCATCTCT TGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGOCTGTTTTA AGAGATTATTCGCACAAACATGCTGGAATGGCAATGCTTGTGAAACCA TTGAAGAAT GT TGGGATCA CGACG CAGAA GCCAGGT TAT CAGC TGATG 10 TTGTAAAA T TT ACC ACAGAGGACAT'G'%ACACAGTGGTCACAATGGTGACAAATGT TGACTIC CCCCAAATCTATC:TATA (SEQ ID NO: 4) The nuceic acid sequence encoding a anan AtRi Ia soluble (extracellular) polypeptide is as follows: l5 ATACTTGGTAGATCAGAAACTCAGGACGTCT T T T T TAATGCTAATT GGGAAAAACACAGAACCAAT CAAACTGGTGTTGAACCGTGT TATGGTG A CAAACATAAAGCCGCAT CT TT TGCTACCTGGAAGAATATTTCTGGT TCCATTGAATAGTCGAAACAAGGT'iTTGGCTCATGATATCAACTGCT AT GACAGGACTGAT TGT GTAG AAAAAAAACA CAGCCCT GAAGTATAT TT 20 TTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTAT T TTCCA CAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCAC c (SEQ ID NO, 5) In certain aspects, the present invention relates to ActRIlb polypeptides. As used herein, the term ActRIlb" refers to a family of aclivn receptor type Ilb (ActRlb) proteins 25 from any species and variants derived from such ActRIlb proteins by mutagenesis or other modification. Reference to ActRIb herein is understood to be a reference to any one of the currently identified forms, Members of the AetR lb family are generally transmembrane proteins, cornposed of a ligandbinding extracelnuar domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonie kinase 30 activity -16- The term "ActR ib polypeptide" includes polypeptides comprising an natural y occurring polypeptide of an ActRb family member as well as any variants thereof (including mutants, fragments, fusions, and pcptidominetic forms) that revan a useful activity. See, for example, WO/2006/0I 2627. For example, ActR-b polypeptides include 5 poypeptides derived from the sequence oa nse known ActRIlb hvn quence at least about 80% identical to the sequence of an ActRIb polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity, For example, an ActRib polypeptide of the ivention may blmd to and inhibit the fiction of an ActR fib protein and/or activin, An ActR lb polypeptide may be selected for activity in promoting red Mood cell fonation in 10 vivo, Examples of Actklib polypeptides include human ActR fIb pre cursor polypeptide (S EQ [D NO: 15) and soluble human ActRIlb polypeptides (eg., SEQ ID NO: 16, 17, 20, and 21). The human ActRflb precursor protein sequence is as fbilows: [NYPA PWVALALLWGSLWPGSGRGEAETRECIYYNANWELERTNQSGLERC SGEQDKRTHHCYASWAj SSGTIELVKKGCWLDDFNCYDRQECVATEENPQ 15 VYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT.LTVLASLL PI. GLSLJIVLLAFW YRHPiKPPYGHVDIHEDPG PPPPSPVCLKPLQL.EIK ARGRFGVWKAQLMNDFVAVRIFPLQDKQSWQS ERE I FSTPGMKH ENLL QFIAAEKRGSNLEVELWL TAFBIDKGSLT DYLKGN I ITWNELCHVAE;TtM SRGLSYLHE DVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLA 20 VRFEPGKPPGDTHGQVGT RRYMAPEVLEGA IN FQ PDA FLt DWYAMGLV LWELVSRCKAADGPVDEYML PFEEEIGQHPSLEEAQUVVVHKKMRPTI 1K. DH WLKH PGLAQLCVT IEECW DR DAEARLSAGCVEEPVS1AIR RSVNGTTS DCLVSLVTSVTNVDLP PKESSI (SEQ ID NO: 15) The signal peptide is single underlined; the extraceIlular domain is in bold and the 25 potential N-inked glycosylation sites are in boxes, The human ActRIlb soluble extracellularr\ processed polypeptide sequence is as follows: SGRGEAETRECIlYNANWELERTNQSGLEPCEGEQDIRLHiCYASWANSS GT IELVKKGCWLDDENCYDPQECVATEEN PQVYFCCCEGNFCNERFTHL 30 PEAGGPEVTY:PPPTAP (SEQ ID NO: 16) The C-terminal "tail of the extracelar domains undefined. The sequeneewth the "tail" deleted (a Al 5 sequence) is as follows: SGR EC AE TREC Yl YANWELE RTNQSGLERCEGEQDKRLCYA W S GCTI EINKKGCCWLUDDENCYDRQECVA TEEN PQVY FCCCEGN FCNER FTMHL 5 EA (SEQ ID NO: 17) The nuclic acid sequence encoding a human ActiRIb precursor ptefis as fellows: (mnleonides 5-1543 of Genbank entry NM_001106) ATCGAC GCCCCTGGGTGGCCCCTC CCCCT CCTCTCGGGGATCGCTGTGCC A CGGCTCTGGG CTGGACCCTAGAt~CGGAG mGCAT CTACTACA 1) CGCCCAAOGGGGAGCGT G GCAGGAACTGCTACCCCT A CTCT GGCCTCGGCCG GAGAGG'CGGTGT AT CAACTctACNEGG:GAGGTT GCAC-AGAAACCCA GTGTTACTT CTCCTG CT GTGAAGCA ACTT TCTGAACGAG' TTCACTC 15 ATT TGCCAGAGCT GGGGGCCCCCGGAAGT2CACCTACGAGCCACCCCA AGCCCCCACCCTCC CC ACiCCCCCCTACCCTCATCTCGACATCCATGACGGACCCTGGCCTCCACC C G TAACGGAATCAAG 20 GGCGGCGCOCT T TGGCT GT GTCTGGAAGCC AGCTCANTAAT GCT TTGTACCTGT'CAAGATCTTCCCACTCCAGGACA(AGCAGTCGTC CAGAG TGAACGGGACATTTCAGCACACCTGGC.ATGAAGCCACAACCT CTA OAT) 'AT GaCCGAGAAGCGAGGCTCC zy' '~ ACCTCGAAG(TAGACTG

GG

1 CTCATCACGGCCT'T CCATGACANGGGCTC.CCTCACGGCAT TA CCTCAA 2$ GGGGCAACATCATCGACAT GGAACGAACTGTGTCATGTACAGACACGAT G TCAC GAGGCCCTCTFCATrACCTCCAT GAGGATGTGCCCTGGTCGCCGTGGCG AGGGCCACAACCOC CTAT TCC CACACGGGACT T TAAAAGTAAGCAATCGT AT TGCTGAAGACGACCTCACAGCGTGCTGGCTGACTTTGGCTTGT GCGATTTGAGC AGGAACCCCA...GGGGtAXACCCACAAGGTAG 30 GCACGAGACGG'TACAT GGCT..CTGAGG ~TCGAGGGAGCCCATCAACT T CCAGAGAGATGCTCGGATTAAGAGCAGGTGT CTGTGGGAGCO OGCCCGAGCTCGCGCCTGT AGTACATGCTGCCCTTTGAGGAAGAGATTGGCCAGCACCCTTCGTTGGA GGAGCTGCAGGAGGTGGGTiCACAAGAAGATGAGCCCCACCATTAAA GATC ACTGOT TGAAACAC COGGCTGGCCOGTTTGTOGACCATCG0 AGGAGTGCTGGGACCATGATGCAGAGGCTCGCTTGTCCGCGGGCTGTGT 5 GGAGGAGCGGGTGTOCCTGATTCGGAGGTOGGTCAACGGCACTACCTCG GACTGTCTCGTTTCCCTGGTGAOCTCTGTCACCAATGTGOAOCGCCCC CT AAAGAGTCAAGCATCTAA (SEQ MD NO: 18) The ucleic acid sequence encoding a human ActRila soluble (extracelular) polypeptide is as f&bows: 10 T C TGGGCGTGGGGAGGCTGAGAACGGGAGTGCATCTACTACAACGCCA ACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTiGAGC TGCGAAGGO CGAGCAGGACAAGGCTGCACTGCTACGOCCTCCTGGGCCAACAGCC GGCACCACGAGCTCGT GAAGAAGGGGT GCTGGCT AGA T 'GACTTCAACT 2 GCTACGATAGGCAGGAG TGT GT GGCCACTGAOGAGAACCCCCAGGTGT A 5 TTT 10GCTGCT G TGAAGGOCAACTTCTGCAACGAGCGCTTCACTCATTT G CCAOAGGCTCGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCC CA CO (SEQ ID NO: 19) In a specific embodiment, the invention relates to soluble Act.R polypeptides. As 20 described herein, the term "soluble ActRil polypeptide generally refers to polypeptides comprising an extracellular domain of an ActR la or ActR b protein. The term "soluble ActRil polypeptide as used herein, includes any naturally occurng extracelular domain of an ActRla or ActR Jib protein as well as any variants thereof includingn mutants, fragments and peptidonime forms) An acti vin-binding ActRil polypeptide is one that retains the 25 ability to bind to activin, including for example, activin AA- AB, BB, or forms that include a C or E subunit. Optional y, an activin-binding ActRil polypeptide wi bind to activin AA with a dissociation constant of I nM or less, The extracelluar domain of an ActIl protein binds to activin and is generally soluble, and thus can be termed a soluble, activin-binding ActRil polypeptide. Examples of soluble, activin -binding ActR a polypeptides include the 30 soluble polypeptides illustrated in SEQ l0N~s: 2, 3, 7, 12 and 13. SEQ ID NO:7 is referred to as A ctRila-h~, and is described father in the Examples Other examples of'soluble, activin-binding ActRUa polypeptides comprise a signal sequence in addition to the 19extracellular domain of an ActR. la protein for example, the honey bee mellitin leader sequence (SEQ ID NO: 8). the tissue plannogen activator (TEA)leader (SEQ ID NO: 9) or ) the native ActRlia leader (SEQ ID NO 10), The ActR1a hFc polypeptide illustrated in SEQ ID NQ:13 uses a TPA leader, Examples of soluble, activinbinding ActRub polypeptides 5 include the soluble polypeptides illustrated in SEQ ID NhOs: 16, 17, 20. Activin-binding ActRub polypeptides may also comprise a signal sequence in addition to the extracellular domain of an ActRUb protein, for example, the honey bee neflitin leader sequence (SEQ ID NO: 8) or the tissue plaminogen activator (TPAj leader (SEQ ID N: 9); Functionally active fragments of ActRlH polypeptides can be obtained by screening 10 polypeptides recombinantly produced from the corresponding fragment of the nucleic acid ectRI polypeptide. addition, ragnnts can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moe or t-Boc chemistry The fragments cm be produced (recombinantly or b cheical synthesis) and tested to identify those peptidyl fragments that can function as antagonistsinhibit) of 15 ActRH protein or signaling mediated by activin. Functionally active varits of ActRI polypeptides can be obtained by screening libraries of modified polypeptides recombinantly produced front the corresponding mutagenized nucleic acids encoding an ActRu polypeptide. The variants can be produced and tested to identify those that can function as antagonists (inhibitors) of ActRil protein or 20 signaling mediated by activin. in certain embodiments, a functional variant of the AtRia polypeptides compares an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs 2 or 3. In certain cases, the functional variant has an amino acid sequence at least 80% 85% 90% 95%, 97% 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3, in certain embodiments, a 25 functional variant of the ActRulb polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 16 or 17, In certain cases. the Functionai variant has an anino acid sequence at least 80% 85%; 90%., 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected fra SEQ ID NOs: 17 or 18 30 Functional variants may be generated by modifying the structure of an ActRil polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g, ex vivo shelf life and resistance to proteolytie degradation in vivo). Such modified ActRlI polypeptides when selected to retain activin binding are considered functional equivalents of ) the naturaly-occuning ActRR polypeptides. Modified AtRil polypeptides can also be produced.. for instanceby amino acid substitution, deletion, or addition. For instance it is . reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a ghamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (eg, conservative mutations) will not have a major effect on the biological activity of the' resulting molecule. Conservative replaements are those that take place within a family of amino acids that are related in their side chains 10 Whether a change in the amino acid sequence of an ActRII polypeptide results n a functional homolog can be readily determined by assessing the ability of the variant ActRI polypeptide to produce a response in cells in a fashion similar to the wildtype ActRII polypeptide In certain embodiments, the present invention contemplates specific mutations of the ActRi polypeptides so as to alter the glycosylation of the polypeptide. Such mutations may 15 be selected so as to introduce or eliminate one or more glycosylation sites, such as Odinked or N-inked glycosylatloa sites Asparagine-l inked glycosylation recognition sites generally comnprise a tripeptide sequence, asparagine hreonine or asparagine~X-serne (where "X" is any anmo acid) which is specifically recognized by appropr ate cellular glycosylation enzymes. The alteration may also be made by the addition of, or substitution by. one or more 20 serine or threonine residues to the sequence of the wild-type ActRIl polypeptide (for 0 linked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (andior amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on an ActRI 25 polypeptide is by chemical or enzymatic coupling of glycosides to the ActRI polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) artgininie and histidine; (b) free carboxyl groups; (c) free s-)fhydryl groups such as those ot cysteine; (d) free h ydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of 30 glutamine. Removal of one or more carbohydrate noieties present on an ActRIl polypeptide may be accorupi shed chemically and/or enzymaticali. Chemical deglycosylation may involve, for example, exposure of the ActR H polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound, This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N~ acetylgalactosaminet while leaving the amino acid sequence intact Enzymatic cleavage of carbohydrate moieties on ActRil polypeptides can be achieved by the use of a variety of 5 endo- and exo-glycosidases as described by Thotakura et at (1987) Meth, Enzymol. 138:350, The sequence of an ActRI I polypeptide may he adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and p cs may Al introduc differing glycosylation patterns that can he affected by the amino acid sequence of the peptide, In general, AetR1I proteins for use in humans may be expressed in a mammalian cell 10 line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be usefi as well This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of an ActRl polypeptide, as well as truncation mutants; poois of combinatorial mutants are especially useful for identifying fumionai variant sequences. The 1S purpose of screening such combinatorial libraries may be to generate, for example, AetII polypeptide variants which bind to activin or other ligands, A variety of screening assays are provided below, and such assays may be used to evaluate variants. For example, an ActRlI variant may be screened for ability to bind to an ActRII igand, to prevent binding of an ActRII ligand to an ActRIl polypeptide or to interfere with signaling caused by 20 an ActRil ligand, The activity of an ActRil polypeptdie or its variants may also be tested in a celdbased or in vivo assay, For example the effect of an ActR polypeptide variant on the expression of genes involved in hematopeiesis may be assessed. This may. as needed, be perfrned in the presence of one or more recombinant ActRi ligand proteins (eg. activin) and cells may 25 be transfected so as to produce an ActRIlI polypeptide and/or variants thereofl and optionally, an ActRil ligand, LAkewise, an ActRil polypeptide may beadninistered to a mouse r other animal, and one or more blood measurements, such as an RBC count, hemoglobin, or reticulocyte count ray b assessed. Comnbinatorially dei ved variants can be generated which have a selective or generally 30 increased potency relative to a naturally occurring ActRIl polypeptide; Likewise mutagenesis can give rise tovants which have intracellular halfives dramatically different than the corresponding a wilddtype ActRil polypeptide. For examplethe altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or otherwise inactivation of a native ActRIl polypeptide Such variants, and the genes which encode then, can be utilized to alter ActR1 5 polypeptide levels by modulating the haiflife of the ActRI polypeptides. For instance, a short half-ife can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant ActRIl polypeptide levels within the cell. In an Fc fusion protein, mutations may be made in the linker (if any) and/or the Fe portion to alter the half-life of the protein" 10 A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ActR II polypeptide sequences For instance, a mixture of synthetic ocigonucleotides can be enzymatically ligated into gene sequences such that the deNenerate set of potential ActRIl polypeptide nucleotide sequences are expressible as individual polypeptides, oralternatively, 15 as a set of larger fusion proteins (e.gor phage display) There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizerand the synthetic genes can then be lighted ino an appropriate vector for expression, The synthesis of degenerate 20 oligonucleotides wel known in the art,e for example Nrang, SA (1983) Tetrahedron 393; Itakura et al,, (1981) Recombinant DNA. Proc. 3rd Cleveland Sympos. Macromolecules. ed, AG Waton Amsterdam: Elsevier pp273>289, Itakura et aL. (l984) Annu. Rev. Biochem, 53:23; Itakura et al, (1984) Science 198:1056; ike et a- (1983) Nucleic Acid Res, 11:477), Such techniques have been employed in the directed evoltion of 25 other proteins (see. fbr exanIle, Scott et aL, (1990) Science 249;386-390; Roberts et aL, (1992) PNAS USA 89-2429-2433; Devilin et aL (1990) Science 249: 404-406; Cwiria et al, (1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409, 5,198,346, and 5,096815). Alternatively, other forms of mutagenesis can be utilized to generate a comubinatorial 30 library. For example, ActR poiypeptide variants can be generated and isolated frorn a library by screening using for example, alanine scanning mta sand thelike (Rufet al., (1994) Biochemistr 3:15611572; Wang et al (1994)J.iot Chem, 269:3095-3099, Balint et al, (1993) Gene 137:109-118;Grodberg aL, (1993) Fur. J Bioche 218:597 601; Nagashima et al, (1993)3ABich Chem. 268:2888-2892; Lowman et al, (1991) Biochemistry 30:10832-10838; and Cunningham et al, (1989) Science 244;10811085), by 5 linker scanning mutagenesis (Gustin et a, (1993) Virology 193:653-660; Brown et aL, (1992) Mo0 Cell Bio 12:2644-2652; McKnight et al, (1982) Science 232:316); by saturation mutagenesis (Meyers t at, (1986) Science 232:613); by PCR mutagenesis (Leung et aL (1989) Method Cei Mol Biol ii 19); or by random rnutaenesls, including chemical rmutagenesis etc. (Miller et al, (1992) A Short Course in Bacterial Genetics, CSRL Press, O Cold Spring Harbor, NY; and Greener et a, (1994) Strategies in Mol ;Biol 7:32-344) Linker scanning mutagenesis paricularly in a combinatorial setting, is an attractive method fbr identifying truncated bioactivee) forms of ActRUI polypeptides A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, arid, for that mater fbr 5 screening IDNA libraries for gene products having a certain property Such techniques will be generally adaptable for rapid screening of fhe gene libraries generated by the combinatorial mutagienesis of ActRil polypeptides. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library ofvectors, and 0 expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encodig the gene whose product was detected, Preferred assays include activin binding assays and activin-mediated cell signaling assays in certain embodiments the ActRIl polypeptides of the invention may further 25 comprise post-translafional modifications in addition to any that are naturally present in the ActRil polyperpides Such modiications include but are not limited torcetylatior, carboxylation, glycosylatiorm phosphorylation, lipidation, and acylation. As a result, the modified ActRRI polypeptides may contain non-amino acid elementssuch as polyethylene glycols lipids, poly- or mono-saccharide and phosphates Effects of such non-amino acid 30 elements on the functionality of an ActRil polypeptide may be tested as described herein for other ActRlR polypeptide variants, When an ActRI H polypeptide is produced in cells by -24 - Cleaving a nascent form of the ActRl polypepide, post-translational processing may also be important for correct folding and/or function of the protein. Different cells (such as CHO, HeLa, MDCK 293, W138 NIHBT or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure S the correct modification and processing of the ActRiI polypeptides. In certain aspects, functional variants or modified forms of the ActRil polypeptides include fusion proteins having at least a portion of the ActRI polypeptides and one or more fusion donains. Well known examples of such fusion domains include, but are not limited to, polyiisidine, Glu-Glu glutathione S transf'erase (GST) hioredoxin, protein A, p C an immunoglobulin heavy ebani constant region (F) malose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For examples some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography, For the purpose of affinity purification revant matrices for affmity chromatography, such as glutathione-, amylase-, and nickel or cobalt- cotjugated 5 resins are used Many of such matrices are available in kit" formsuch as the Pharmacia GST purification system and the QlAexpresssystem (Qiagen) useful with (HiS 6 ) fusion partners. As another example a fusion domain may be selected so as to facilitate detection of the ActRH polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g, GFP) as well as "epitope tags, which are usually short peptide sequences for 0 which a specific antibody is available Well known epitope tags for which specific monoclonal antibodies are readily available include FLA G influenza virus haemagglutinin (HA). and c-mye tags. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefro. The liberated proteins can 25 then be isolated from the fusion domain by subsequent chromatographic separation in certain preferred embodiments an ActRil polypeptide is fused with a domain that stabilizes the ActR'l polypeptide in vivo (a "stabilizer" domain) By "stabilizing? is mcant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinietic effect Fusions with the Fc 30 portion of an immunoglobulin are known to confer desirable pharmacokineic properties on a wide range of proteins Likewise fusions to human serumalbumin can corner desiable properties, Other types of fusion domains that may be selected include multimerizing (e g, dimezing. tetramerizi ng) domains and functional domains (that confer an additional biological fUnction, such as further stimulation of mmsle growth). As a specific example, the present invention provides a fusion protein comprising a soluble extracelular domain ofActlla fused to an Fe domain (eig. SEQ ID NO: 6. 5 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT EVTVV (A)VSHEDPEVKFNWYVDG VEVKNAKTKPREEQYNSTYRVVSV 4TV >LQDWTNGKEYKCK ()SNKALPVPIEKTISKAK GQPREPQVYTTLFPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG PFFLYSKLVDKSRWQQGNVFSCSVMHEALHN (A) HYTQKSLSL SPGKK As an additional specific example, the present invention provides a fusion protein D comprisng a soluble extracelular domain of ActRllb fused to an Fe domain (e.g., SEQ ID NO: 21 SGPGEAETPEGIYYNANNWELERTMQSGLERCEGEQDKRLH CYASWANSSGTIELKKGCWLD DFNCYDRQECVATSENPVY FC1CEG FCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCP PCPAPELLGGPSVF FPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK'T S KPREEQYNSTYVVSVLTVHQDWLNGKYYKCKVSNKALPVPTEKTISKAKGQPREPQVYTL PPSHEEMTKNQVSLTCLVKGFY PS DIAVEWESNGQPENNYKTTPPVLDSDGS FPLYSKLTVD KSRWQQGNVFSCSVMHEAL HN HYTQKSLSLSPGK Optionally, the Fc domain has one or more mutations at residues such as Asp -265 lysine 322,and Asn-434 in certain cases, the mutant Fc domain having one or more ofthese 0 mutations (eg. Asp-265 mutation) has reduced ability of binding to the Fey receptor relative to a widtype Sc donman in other cases, the mutant cf domain having one or more of these mutations (eg., Asn-434 mutation) has increasedability of binding to the M-IC class I related Fc-receptor (FcRN) relative to a wildtype Fc domain. It is understood that different elements of the fusion proteins may be arranged in any 25 manner that is consistent with the desired functionality. For example, an ActRil polypeptide may be placed C-terminal to a heterologous domain oralternatively, a heterologous domain may be placed C-terminalto an Act~ polypeptide. The ActRil polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or Nterminal to either domain or between the 30 domains. -26- In certain embodiments, the ActRI polypeptides ofthe present invention contain one or more modifications that are capable of stabili-ing the Act[il polypeptides. For example, ) such modifications enhance the in vitro half lifi of the ActRR polypeptides. enhance circulatory half life of the ActRi I polypeptides or reducing proteolytic degradation of the 5 ActRIT polypeptides. Such stabilizing modifications include, but are not limited to fusion proteins (including, for exampefusiori prOteins comprising an ActRRl polypeptide and a stabilizer domain), modtations of a glycosylation site (including for example, addition of a glycosylation site to an ActRIl polypeptide, and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from an ActR If polypeptide) As 10 used herein, the term "stabilizer domain not only refers to'-fusion domaineg, Fe) as in the case of fusion proteins, but also includes nonproteinaceous rmodifications such as a carbohydrate moiety, or nonproteinaceous moiety, such as polyethylene glycol, uncertain embodiments, the present invention makes available isolated and/or purified forms of the ActR polypeptides. which are isolated from. or otherwise substantially free of 15 other proteins, AczR I polypeptides will general be produced by expression from recombinant nucleic acids. 3. Nucleic Acids EnodinAeLIPLyeptides In certain aspects, the invention provides isolated and/or recombinant nucleic acids 20 encoding any of the ActRII polypeptides (eg soluble ActRlla polypeptides and soluble ActRIlb polypeptides), including fragmens, functional variants and fusion proteins disclosed herein. For example, SEQ ID NO; 4 encodes the naturally occurring human ActRila precursor polypeptide, while SEQ ID NO: 5 encodes the processed extracellular domain of At tRila For example, SEQ ID NO: 18 encodes the naturally occurring human ActRbll 25 precursor polypeptide while SEQ ID NO: 19 encodes the processed extracellular domain of ActRIlb. The subject nucleic acids may be singlestranded or double stranded. Such nucleic acids may be DNA or RNA molecules These nuclee acids may be used. for example, in methods for making ActR H polypeptides or as direct therapeutic agents (e.g, in a gene therapy approach). 30 In certain aspectsthe subject nuclei acids encoding ActRila polypeptides are further understood to includenucleic acids that are variants of SEQ ID NO: 4 or 5 In certain aspects, the subject trucleic acids encoding ActRilib polypeptides are rather understood to include nucleic acids that are variants of SEQ ID NO: 18 or 19. Variant nucleotide sequences S include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants. 5 In certain embodiments, the invention provides soiated or recombmaiat nucleic acid sequences that are at least 80%. 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID1 NOs: 4, 5, 18 or 19. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NOs: 4, 5, 18, or 19, and variants of SEQ ID NOs:4, 5, 18 or 19 are also within the scope of ths invention, in further embodiments, the nucleic acid 10 sequences of the invention can be isolated, recombinant, and/or fused with a heterologous nucleotide sequenceor in a DNA library, In other embodiments, nucleic acids of the invention also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NOs: 4. 5, 18, or 19, complement sequence of SEQ ID NOs: 4. 5. 18, 15 or 19. or fragments thereof. As discussed above, one of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied, One of ordinary skill in the awill understand readily Tat appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6,0 x sodium codeodium citrate ( at about 45 'C, 20 -lblowed by a wash of 2 0 x SSC at 50 *C, For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 'C to a high stringency of about 0.2 x SSC at 50 C.n addition the temperature in the wash 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 may be varied, or temperature or salt concentration 25 may be held constant while the other variable is changed. in one embodimentthe invention provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2x SSCa at room temperature. Isolated nucleic acids which differ from the nudeic acids as set forth in SEQ 11) NOs: 4 5 18, or 19 due to egen in the genetc code are also within the scope of the 30 invention. For example a number of arnino acids are designated by more ti-ian one triplet, Codons that specify the same amino acid, or synonyms (for example, CAU and GAC are 28 synonyms for histidine) may result in "silent"mutations which do not affect the amino acid sequence of the protein. However t is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. Onekilled in the art will appreciate that these variations in one or more 5 nucleotides (up to about 3-5% of the nucleotides) of the nuclei acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. h- certain embodiments, the recombinant nucleic acids of the invention may be 10 operablylinked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nud'eotide sequences will generally be appropriate to the host cell used fr expression, Numerous types of appropriate expression vectorsand suitable regulatory sequences are known in the art for a variety of host cells, Typically said one or mor regulatory nucleotide sequences may include, but are not limited to, promoter sequences, 15 leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurtng promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a 20 cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell usedt In certain aspects of the invention, the subject nucleic acid is provided in an 25 expression vector comprising a nucleotide sequence encoding an ActR1l polypeptide and operablyhinked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the ARctI< polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements, Exemplary regulatory sequences are described in Goeddel; Gene &pression Technoogy: 30 Methods in Evymdogi Acade-nic Press. San Diego CA (1990) For instance any of a wide variety of expression control sequences that control the expression of a DNA sequence when -29operatively linked to it may be used in these vectors to express DNA sequences encoding an ActRl polypeptide, Such useful expression control sequences., include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 5 prornoter whose expression is directed by T7 RNA polymerase, the major operator and prornoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphogiycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, eg, Pho5, the promoters of the yeast c-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of 10 prokaryotic or eukaryotic cells or their viruses, and various combinations thereof It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transforned and/or the type of protein desired to be expressed, Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also 15 be considered. A recombinant nucleic acid of the invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant ActRil polypeptide include plasmids and other vectors For 20 instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL derived plasmnids, pEX-derived plasmids, pBTac-derived plasmids and pU C-derived plasmids for expression in prokaryotic cells, such as E. coli. Some mammalian expression vectors contain both prokaryotic segluences to facilitate the propagation of the vector in bacterial and one or more eukaryotic transcription units that 25 are expressed in eukaryotic celis The pcDNA/1amop, pcDNAI/nee pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRS eo, pMSc, pSVT", pko-neo and pIyg derived vectors are examples of mannialian expression vectors suitable for transition of eukarvotic cells. Some of these vectors are modified with sequences from bacteria plasmids, such as pBR322 to facilitate repication and drug resistance selection in both prokaryotic and eukaryotic cells 30 Alternatively, derivatives of viruses such as the bovine papilioia virus (BPV-1)l or Epstein Barr virus (pHEBo, pREP-derived and p 2 0 5 ) can be used for transient expression of proteins in eukaryotic cells, Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods ) employed in the preparation of the plasmids and in transformation of host organisms are well known in the art., For other suitable expression systems for both prokaryotic and eukaryotic 5 cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 3rd Ed., ed, by Sambrook, Fritsch and Maniatis (Cold Sprng Harbor Laboratory Press, 2001), in some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pV L-derived vectors (such as pVL1.392, pVL1393 and pVL941), 10 pAcUW-derived vectors (such asp) and pBlue3a-derived vectors (such as the 1-gal containing pBlueBac 111), in a preferred embodiment, a vector will be designed for production of the subject ActfRIl polypeptides in C-O0 cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen, Carlsbad, Calif) and pCIeo vectors (Promega, Madson, 15 Wise.) As will be apparent, the subject gene constructs can be used to cause expression of the subject ActRil polypeptides in cells propagated in culture, e~g, to produce proteins, includ ng fusion Proteins or variant proteins for purification. This disclosure also pertains to a host cell transfeted with a recombinant gene including a coding sequence (e g SEQ ID NO 4, 5, 18, or 19) for one or more of the subject 20 ActRil polypeptides. The host cell may be any prokaryotic or eukaryotic cel For example, an ActRil polypeptide of the invention may be expressed in bacterial cells such as .E coU, insect cells (e~g using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. Accordingly, the present invention further pertains to methods of producing the 25 subject Actkil polypeptides. For example, a host cell transfected with an expression vector encoding an ActRlla or ActRub polypeptide can be cultured under appropriate conditions to allow expression of the ActR1I polypeptide to occur. The ActRIl polypeptide may be secreted and isolated from a mixture of cells and medium containing the ActRl polypeptide Alternativelythe ActRIl polypeptide may be retained cytoplasmically or in a membrane 30 fraction and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culure are well known in the art.

The subject ActRil polypeptides can be isolated from cell culture medium, host cells, or both. usimg techniques known in the art fbr purifying proteins, including Ioexchange ) chromatography, gel filtration chromatography, ultrafiltration, el ectrophoresis, immunoaffinity purification with antibodies specific fbr particular epitopes of the ActRIl 5 polypeptides and affinity purification with an agent that binds to a domain fused to the ActRi polypeptide (e.g., a protein A column may be used to purify an ActRiia-Fc or ActRllb-E fusion). In a preferred embodiment, the ActR II polypeptide is a fusion protein containing a domain which facilitates its purification, In a preferred embodiment, purification is achieved by a series of column chromatography steps, including, for example, 10 three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenyisepharose chromatography. size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. As demonstrated herein, ActRila-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS 1 PAGE. This level of purity was sufficient to achieve desirable results in mice, rats and non humnan primates. In another embodiment, a fusion gene coding for a purification leader sequence, such as a polykHis)/enterokinase cleavage site sequence at the Nterminus ofthe desired portion of the recombinant Actkil polypeptide, can allow purification of the expressed fusion protein 20 by affinity chromatography using a Ni 2 " metal resind The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ActR1I polypeptide (eg, see Hochuli et al, I 9 . hromatography 411 77 and Janknecht et al, PN AS USA 88:8992) Techniques for making fusion genes are well known. Essentially, the joining of 25 various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended terming for ligation. restriction enzyme digestion to provide for appropriate terniin, fillingin of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by 30 conventional techniques including automated DNA synthesizer, Alternatively, PCR amplification of gene framents can be carried ot uing ancoir primers which give rise to 32complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chineric gene sequence (see, for example, Currentprotocols in ) Molecular Biology eds Ausubel et at., John Wiley & Sons: 1992) 5 4. A at Activin and ActRU Antagonists The data presented herein demonstrates that antagonists of active n-ActR i signaling can be used to increase red bWod cell or hemoglobin levels, Although soluble ActR lla and ActRulb pol ypeptides, and particularly ActR la-Fe and ActRlib-c, are preferred antagonists, and although such antagonists may affect red blood cell levels through a mechanism other 10 than activin aag (e g. activin inhibition may be an indicator of the tendency of an agent to inhibit the activities of a spectrum of molecules, including, perhaps, other members of th.e TGF-beta superfamily and such collective inhibition may lead to the desired effet on hematopoiesis), other types of activin-ActRI antagonists are expected to be useful, including anti-activin (eg, activin A pr c and ) antibodies anti-ActRila antibodies, anti-ActRilb 15 antibodies, antisense, RNAi or ibozyme nuceic acids that inhibit the production of ActRila aid/or ActRIub, and other inhibitors of activin, ActR lb or ActRiHa, particularly those that disrupt activin-ActRI la and/or activin-ActRfib binding. An antibody that is specifically reactive with an ActRIl polypeptide(ega soluble ActRila or ActRflb polypeptide) and which either binds competitively to ligand with the 20 ActR il polypeptide or otherwise inhibits ActRIT-mediated signaling may be used as an antagonist of AcetRil polypeptide activities. Likewise, an antibody that is specifcaly reactive with an actvin P, pc Pc or -polypeptide or any heterodimer thereof, and which disrupts ActRia and/or ActR1lb binding may be used as an antagonist By using immunogens derived from an ActRIla polypeptide AetRlb polypeptide or 25 an activin polypeptide, and-protein/anti-peptde antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: i 988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the activin, ActRila or ActRllb polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a 30 fusion protein Techniques for conferring immunogenicity on a protein or peptide include conjugatior to careers or other techniques well known in the art. An immunogenc portion of - 33 an ActRtl; or activin polypeptide can be adunistered in the presence of adjuvant, The progress of inmunization can be monitored by detection of antibody titers in plasma or serm. Standard ELISA or other imunnoassays can be used with the imnunogen as antigen to assess the levels of antibodies. Following immunization of an animal with an antigenic preparation of an activin, ActRlia or ActRilb polypeptide, antisera can be obtained and. if desired, polyclonal antibodies can be isolated from the serum. To produce nonoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by Mandard somatic cell fusion procedures with immortalizing cells such as myeloma cells to D yield hybridomia cells. Such techniques are well known in the art, and include, for example the hybridona technique (originally developed by Kohler and Milstein, (1975) Nature,256. 495-497), the human B cell hybridoma technique (Kozbar et al (1983) Immunology Today, 4: 72), and the EBVVhybridoma technique to produce human monocional antibodies (Cole et al, (1985) Monoclonal Antibodies and Cancer TherapyAlan P. Liss, inc pp 77-96), 5 Hybridora cels can be screened irmunochemically for production of antibodies specifically reactive with an activii, ActRlla or ActRb polypeptide and rnonocional antibodies isolated from a culture comprising such hybridoma cells. The term "antibody" as used herein is intended to include whole antibodies, eg., of any isotype (IgG, igA, 1gM, IgE etc) and includes fragments or domains of 0 immunoglobulins which are reactive with a selected antigen. Antibodies can be fagmented using conventional techniques and the fragments screened for utility and/or interaction with a specific epitope of interest Thus, the term includes segments of proteolfyticaly-leaved or recombinantlyprepared portions of an antibody molecule thai are capable of selectively reacting with a certain proteinNon-imiting examples of such proteolytic and/or recombinant 25 fragments include Fab, F(ab)2, Fab' Fv, ard single chain antibodies (scfv) containing a NL] and/or V[-] domain joined by a peptide linker, The seFvs may be covalenty or non covalently linked to form antibodies having two or more binding sites. The term antibody also incides polyclonal, mnonoclonal o other purified preparations of antibodies and recombinant antibodies, The term recombinant antibody", means an antibody, or anigen 30 binding domain of an nmunoglobulin, expressed from a nucleic acid that has been constructed using the techniques of molecular biologyuch as a humanized antibody or a - 34..

fully humannitibody developed from a single chain antibody, Single domain and single chain antibodies are also included within the term >recombinant antibody". In certain embody s antibody of the invention is a monoclonal antibody. and in certain erbod iments. the invention makes available methods for generating novel antibodies, 5 For example, a method for generating a monocional antibody that minds specifically to an ActRlla polypeptidee, ctRib polypeptide, or activin polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polyp eptide effective to stimulate a detectable immune response, obtaining antibody producing ceis (eg, cells from the spleen) from the mouse and fusing the antibody d producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the antigen. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the antigen. The monoclonal 5 antibody may be purified from the cell culture, The adjective "specifically reactive wit , as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g. an activin, ActRila or ActRllb polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, 0 detecting the presence of the antigen of interest in a particular type of biological sample In certain methods employing the antibody, such as therapeutic application, a higher degree of specificity in binding mray be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and crossreacting polypeptides. One characteristic that influences the 25 specificity of an antibody:antigen interaction is the affnity of the anti body for the anti gen, Although the desired specificity may be reached with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about 10t 10" 10" 0 M or less, In addition, the techniques used to screen antibodies in order to identify a desirable 30 antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding A 35 .

variety of different techniques are available fr testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (eg, the Biacor binding assay, Biacore AB, Uppsala, Sweden) sandwich assays (e.g., the parmagnetic bead system of IGEN 5 international Ic, Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunoh istochern istry Examples of categories of nucleic acid compounds that are activir or ActRil antagonists include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nucleic acid compound may be single or double stranded. A double stranded 10 compound may also include regions of overhang or non-'omplementaritywhere one or the other of the strands is single stranded. A single stranded compound may include regions of self-cmplementari meaning that the compound forms a so-called "hairpin" or "stemloop" structure, with a region of double helical structure. A nuclei acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no 15 more than 500, no more than 250, no more than 100. or no more than 50, 35. 25, 22, 20 IS or I rnucleotides of the fillength ActRil nucleic acid sequence or activin $iA. @Ja, or nucleic acid sequence. The region of eomplementarity will preferably be at least 8 nucleotides, and optionally about 18 to 35 nucleotides, A region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as 20 the coding sequence portion, Generally, a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in lngth, and optional tlent i be about 14 to about 50 nucleotides, A nucleic acid may be a DNA (particularly for use as an antisense), RNA or RiNA;DNA hybrid Any one strand may include a mixture of DNA and RNA, as wel as modified forms that cannot readily be classified as either DNA or RNA, Likevise a 25 double stranded compound may be DNA;DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA as well as modified firms that cannot readily be classified as either DNA or RNA. A nucleic acid compound may include any of a variety of modifications including one or modifications to the backbone (the sugar-phosphate portion in a natu nucleic acid, including internucleotide linkages) or the base portion (the 30 purine or pyrimidine portion of a natural nuclei acid) An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to prove characteristics such as stability in the serun, in a cell or in 36a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds, In the case of an RNAi p construct, the strand complementary to the target transcript will genally be RNA or modifications thereof The other strand may be RNA, DNA or any other variOn. The 5S duplex portion of double stranded or single stranded "hairpin" RNAi construct will generally have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymes orDNA enzymes and may also contain modified frms Nucleic acid compounds may inhibit expression of the target by about 50%, 75%C 90% ormore when contacted wh 0 cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 nicromolar, Nucleic acid compounds may also be tested for effects on, for example, red blood cell levels, 5 5, ScreeningAssays In certain aspects; the present invention relates to the use of ActRtfl polypeptides (e.g soluble ActR lIa or ActRi b polypeptides) and activin polypeptides to identify compounds (agents) which are agonist or antagonists of the activin-ActRITa and/or activinActitb signaling pathway Compounds identified through this screening can be tested to assess their 0 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. There are numerous approaches to screening for therapeutic agents for increasing red blood cell or hemoglobin levels by targeting activin and ActR11 signaling. In certain embodiments, high-throughput screening of compounds can be carried out to identify agents 25 that perturb activin or ActRil-mediated effects on a selected cell lire In certain embodiments, the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an ActRIla or ActRIlb polypeptide to activin, Alteatively, the assay can be used to identify compounds that enhance binding of an ActRla or ActR lib polypeptide to activin. In a further embodment, the compoundscan be identified by their 30 ability to interact with an activin, ActRilb polypeptide, or Actilla polypeptide, A variety of assay fo-mats will suffice and in light of the present disclosurethose not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. Asdescribed herein, the test compounds (agents) of the invention may be created by any comnbinatorial chemical method, Alternatively, the subject compounds may be naturally 5 occurring biomolecules synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced. for example, by bacteria, yeast, plants or other organisms (eig., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantdy. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, 0 polypeptides, peptidormimetics, sugars, hormones, and nucleic acid moleules In a specific embodiment the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daitons. The test compounds of the invention can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry. These 5 libraries can comprise, for examplalcohols aikyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in inial screening steps Optionally, the compounds may be optionally derivatized with other compounds and have derivatizing groups that faciliate isolation of the compounds, Non 0 limiting examples of' devatizing groups include biotin, fluorescein. digoxygenin, green fluorescent protein, isotopes, pilyhi stidine, magnetic beads gutathione S transbrase (GST), photoactivatible crossainkers or any combinations thereof In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds 25 surveyed in a given period of time. Assays which are performed in cellfree systems, such as may be derived with purified or semi-purified proteins, are often preferred as 'primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound, Moreover, the effects of cedar toxicityor bioavilability of the test compound can be 30 generally ignored in the in vitro system, the assay instead being focused pinarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding :38 affinity between an ActRila polypeptide and activin and/or between an ActRiUb polypeptide and activin Merely to llustrate, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified ActRila polypeptide which is ordinarily capable of binding to activin, To the mixture of the compound and Ac:R~la polypeptide is then added a composition containing an AcRlia ligand, Detection and quantification of ActR ila/activin complexes provides a means for determining the coTmpound's efficacy at inhibiting (or potentiating) complex tomation between the ActRiTa polypeptide and activin. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline fir comparison. For example, in a control assay, isolated and purified activin is added to a composition containing the ActRlfa polypeptide, and the formation of ActRila/activin complex is quantitated in the absence of the test compound, It will be understood that in general, the order in which the 5 reactants may be admixed can be varied, and can be admixed simultaneously. Moreover, in place of purified proteins, cellular extracts and lysates may be used to render a suitable cell free assay system. Compounds that affect ActRI~b signaling may be identified m0 a similar manner using an ActR ilb polypeptide and an ActRillb ligand, Complex formation between the ActRIH polypeptide and acti vin may be detected by a ) variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (e.g, 2P, 35S "C or 3 H). fluorescently labeled (e.g, FITC), or enzymatically labeled AetRila or ActRI1b polypeptide or activin, by inmunoassay, or by chromatographic detection, in certain embodiments, the present invention contemplates the use of fluorescence ?5 polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of mteraction between an ActR I polypeptide and its binding protein. Further, other modes of detection, such as those based on optical waveguides (PCT Publication WO 96/26432 and US, Pat, No. 5,677,196), surface plasmnon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with 30 many embodiments of the invention.

Moreover, the present in vention contemplates the use of an interaction trap assay, also known as the "two hybrid assay," for identifying agents that disrupt or potentiate interaction between an ActRil polypeptide and its binding protein, See for example, U.S, Pat. No. 5,283,317; Zervos et at (1993) Cell 72:223-232; Madura et aL (1993) J Biol Chern 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and lwabuchi et a. (1993) Oncogene 8:1693~1696), In a specific embodiment, the present invention contemplates the use of reverse two hybrid systems to identify compounds (e.g. small molecules or peptides) that dissociate interactons between an ActR11 polypeptide and its binding protein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and 11.S. Pat. Nos, 5,525,490; 55280; and 5,965,368. In certain embodiments, the subject compounds are identified by their ability to interact with an ActR l or activin polypeptide of the invention, The interaction between the conipound and the ActRita, ActR llb, 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 photoscrossiinking. radliolabeled ligand binding, and afinity chromatography (Jakoby WB et al 1974, Methods in Enzymology 46: 1). In certain cases. the compounds may be screened in a mechanism based assay, such as an assay to detect compounds which bind to an activan or ActRil polypeptide. This may include a solid phase or fluid phase binding event. Alternatively, the gene encoding an activin or ActRil polypeptide can be transfected with a reporter system (e. ggaiactosidase luciferase or greoen fluorescetnto cell and screened against the library op ona y by a high throughput screening or with individual members of the library. Other mechanism based binding assaymay be used, for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well bead or chip or 5 captured by an immobilzed antibody or resolved by capillary electrophoresisT 1he bound compounds may be detected usually using colorimetric or fluorescence or surface plasmon resonance, 6. a rapeutic Uses so in certain embodiments, activir/ActRI antagonists (e g. ActRU or ActRib polypeptides) of the present invention can be used to increase red blood cell levels in 40, marmmals such as rodents and primates, and particularly human patients, In certain embodiments, the present invention provides methods of treating or preventing anemia in an individual in need thereof by administering to the individual a therapeutically effiective amount of an activinActRla antagonist, such as an ActRita polypeptide, or a therapeutically s effective amount of an activin-AcRemb antagonist, such as an ActRfb polypeptide, In certain embodiments, the present invention provides methods of promoting red blood cell formation in an individual by administering to the individual a therapeuticaly effective amount of an activin-ActR l antagonist, particularly an ActRiH polypeptide. These methods may be used for therapeutic and prophylactic treatments of mammals, and particularly 10 humans. As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated 15 control sample. The term "treating" as used herein includes prophylaxis of the named condition or ameioration or elimination of the condition once it has been established In either case prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent. 20 As shown herein, activin-ActRila antagonists and activin-AetRib antagonists may be used to increase red blood cell, hemoglobin or reticulocyte levels in healthy individuals, and such antagonists may be used in selected patient populations. Examples of appropriate patient populations include those with undesirably low red blood cel or hemoglobin levels, such as patients having an anemia, and those that are at risk for developing undesirably low 25 red blood cel or hemoglobinleves such as those patients that are about to undergo major surgery or other procedures that may result in substantial blood loss, in one embodiment, a patient with adequate red blood cel level is treated with an activin-ActRila antagonist to increase red blood cell levels, and then blood is drawn and stored fbr later use in transfusions, I one embodiment, a patient with adequate red blood ceil levels is treated with an activin 30 ActRub antagonist to increase red blood cell levels, and then blood is drawn and stored for ater use in transfusions.

Activin-ActR II antagonists disclosed herein, and particular ActRilae and ActRIlb proteins, nay be used to increase red blood elB levels in patients having an anemia. When ) observing hemoglobin levels in humans, a level of less than nonnal for the appropriate age and gender category may be inMcative of anemiaalthough individual variations are taken 5 into account. For example, a hemoglobin level of 12 g/di is general considered the lower limit of normal in the general adult population Potential causes include blood-loss, nutritional deficits, medication reaction, various problems with the bone marrow and many diseases. More particularly, anemia has been associated with a variety of disorders that include, for example, chronic renal failure, ny elodyspiastic syndrome, rheumatoid arthritis, 10 bone marrow transplantation. Anemia may also be associatedwith the following conditions: solid tumors (e.g. breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (e'g. chronic lymphocyte leukemia non-lodgiQns and Hodgkmis lymphomas); tumors of the hematopoietic system (eg. leukemia, myelodysplastic syndrome, multiple ryeka radiation therapy; chemotherapy (e.g. platinum containing regimens); inflanmnatory and 15 autoimmune diseases, including, but not limited to, rheumatoid arthrtis, other inflammatory arthritides, systemic lupus erythematosis (SLE), acute or chronic skin diseases (eg. psoriasis), inflammatory bowel disease (e.g. Crobrfs disease and ulcerative colitis); acute or chronic renal disease or failure including idiopathic or congenital conditions; acute or chronic liver disease; acute or chronic bleeding; situations where transfusion of red blood cells is not 20 possible due to patient allo- or auto-antibodies and/or tor religious reasons (e,g, some Jehovah's Witnesses); infections (e g. malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell disease, thalassemias; drug use or abuse, e.g, alcohol misuse; pediatric patients with anemia from ny cause to avoid transfusion; and early a s or patients with Underlying cardiopulmonary disease with arena who cannot receive 25 transfusions due to concerns about circulatory oveloaad. Patients may be treated with a dosing regimen intended to restore the patient to a target hemoglobin level, usually between about 10 g/dl and abou1125 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 henatocrit 30 levels (percentage of the volume of a blood sample occupied by the cels) can be used as a measure for the condition of red blood cells. leniatocrit levels f"r healthy individuals range from 41 to 51% for adult males and from 35 to 45% for adult males Target heiatocrit levelare usually around 30-33%. Moreover, hemoglobinlhematocrit levels vary fromn person to person, Thus, otimally, the target hemogiobin/hematocrit level can be ) individualized for each patient. The rapid effect on red blood cell levels of the activin-ActRi a antagonists disclosed 5 herein indicate that these agents act by a different mechanismthan Epo. Accordingly these antagonists may be useful for increasing red blood cell and hemoglobin levels in patients that do not respond weR to Epo For example,a advi-ActRlia at smay be beneficial for a patient in which administering of a normal to increased (>300 IU/kg/week) dose of Epo does not result in the increase of hemoglobin level up to the target leve Patients with an .0 inadequate Epo response are found for all types of anemia, but higher numbers of non responders have been observed particularly frequently in patients with cancers and patients wvith end-stage renal disease An inadequate response to Epo can be either constitutive (ie observed upon the first treatment with Epo) or acquired (eg. observed upon repeated treatment with Epo). 15 The activinActR Iantagonists may also be used to treat patients that are susceptible to adverse effects of Epo. The primary adverse effects of Epo are an excessive increase in the hematocrit or hemoglobinefvels and polycythemia. Elevated hematocrit levels can lead to hypertension (more particularly aggravation of hypertension) and vascular thrombosis. Other adverse effects of Epo which have been reported, some of which related to hypertension, are 20 headaches. influenza like syndrome, obstruction ofshunts, myocardial infarctions and cerebral convulsions due to thrombosis, hypertensive encephalopathy, and red cell blood cell applasia (Singibarti, (1994) AO Clin Investig 72(suppl 6), S36-S43; H ort et at. (2000) Nephrol Dial Transplant i 5(suppl 4), 51-56; Delanty et at (1997) Neurology 49, 686-689; Bunn (2002) N EngI3 Med 346(7), 522-523), 25 Pharmaceutical Cfomgpositions in certainembodimentsactivin-ActR1 antagonists (g., ActRila and ActRllb polypeptides) of the present nvertion are formulated with a pharmaceutically a ptabie carrier. For example, an ActR I polypeptide can be administered alone or as a component of 30 a pharmaceutical formulation therapeuticc composition). The subject compounds may be - 43.formulated for administration in any convenient way for use in human or veterinary medicine; In certain embodiments, the therapeutic method of the invention includes administering the composition systemicalig or locally as an implant or device. When 5 administered, the therapeutic composition for use in this invention is, of course, in a pyrogen free, physiologically acceptable form. Therapeutically useful agents other than the activin ActRilantagonists which may also optionally be included in the composition as described above, nmy be administered simultaneous or sequentially with the subject compounds (esg, ActR!Ila and ActRib pol ypeptides) in the methods of the invention. 0 typically, activin-ActRil antagonists will be administered parenterally. Pharmaceutical compositions suitable for parenteral administration may comprise one or iore ActR iI polypeptides in combination with one or more phanmaceutically acceptable sterile isotonic aqueous or nonaqueous soutions. dispersions. suspensions or em ulsions or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just 5 prior to use which may contain antioxidants, buffers, bacterostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agent ExamUples of suitable aqueous and nonaqueous canriers which may be ernployed in the pharmaceutica compositions of the invention icle water, ethanolpolyols (such as glycerol, propylene glyc polyethylene glycol, and the like), and suitable mixtures thereof 0 vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions. and by the use of suarfaetants. Further, the composition may be encapsulated or injected in a form for delivery to a R5 target tissue site (eg. bone marrow). In certain embodiments, compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g, ActRila or ActAllb polypeptides) to a target tissue site (eg., bone marrow) providing a structure for the developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow release of the ActRI polypeptides, Such matrices 0 may be formed of materials presently in use for other implanted medical applications, - 44- The choice of matnx material is based on biocompatibilitybiodegradabi ity mechanical properties, cosmetic appearance and interftaeproperties. The particu ar p application of the subject compositions will define the appropriate formnlation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate 5 tricalciumphosphate, hydroxyapati te, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are non-iodegradable and cheically defined, such as sintered hydroxyapatite, bioglass' aluminates, or other ceramics. Matrices may be comprised of 0 combinations of any of the above mentioned types of material such as polylactic acid and hydroxyapati tt or collagen and tricalciumphosphate. The biocerami~cs mnay be altered in composition, such as in caicium-aluminaterphosphate and processing to alter pore size, particle size, particle shape, and biodegradabiliy. in certain embodiments, methods of the invention can be admninistered for orally, eg, 5 in the form of capsules, cachets, pills. tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, o as a solution or a suspension in an aqueous or nonlaquenus iquid, or as an oilin-water or water-in-oil liquid emulsion, or as an xror syrup, or as pastilles (using an inert base. such as gelatin and glycerin. or surose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an 0 agent as an active ingredient, An agent may also be administered as a bolus, electuary or paste, In solid dosage fomns for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceuticals acceptable carriers, such as sodium citrate 25 or dicalcium phosphate and/or any of the following: (1I) fillers or extenders, such as starches, lactose suosese glucose mannitol, and/or silicic acid; (2) binders, such as, for example' carboxymethylcellose, alginates; gelatin polyvinyl pyrrolidone sucrose, and/or acacia; (3) humectants, such as glycerol (4) disintegrating agents, such as agar-agar calcium carbonate, Potato or iapioca starch, d, certain suicates, and sodium carbonate; (5) solution 30 retarding agents; such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol -45 onostearate;(8) absorbentsi such as kaolin and bentonite clay; (9) lubricants such a taic, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof and (10) colorng agents. in the case of capsules, tablets and pills, the pharmaceutical compositions may also compose buying agents. Solid compositions of a 5 similar type may also be employed as filers in soft and hardilled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emuions.microenmulsionssolutions, suspensions. syrups and elixirs, In addition to the 0 active inredient the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol. isopropyl alcohol, ethyl carbonatethl acet benzyl alcohol, benzyl benzoate, propylene glycol. l,3butylene glycol, oils (in particular, cottonseed groundnut, corn, germ olive castor, and sesame oils) glycerol, tetrahydrofuryl alcohol polyethylene glycols and fatty acid 5 esters of sorbitan, and mixtures thereof Besides inert diuents, the oral compositions can also include adjuvants such as wetting agents enusifying and suspending agents sweetening, flavoring, coloring, perfuming, and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyox yethyene sorbitol and sorbitan esters. O microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of nicoorganisms may be ensured by the inclusion of various antibacterial and antifungal 25 agents, fBor example, paraben, chorobutanol, phenol sorbic acid, and the like, tmay also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, In addition, prolonged absorption of the injecable pharmaceutical form may be brought about by the inclusion of agents which delay absorption. such as aluminum monostearate and gelatin. 30 it is understood that the dosage regimen will be determined by he attending physician considering various factors which modify the action of the subject compounds of the 46invention (eg, ActRla and ActfRIib polypeptides). The various factors include but are not limited to, the patient's red blood cell count, henioglobin level or other diagnostic assessments, the desired target red blood cell count, the patients age, sex, and diet, the severity of any disease that may be contributing to a depressed red blood cell level, time of 5 administration, and other clinical factors. The addition of other known gowth factors to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of red blood cell and hemoglobin levels, as well as assessments of reticulocyte levels and other indicators of the hematopoietir process, Experiments with primates and humans have demonstrated that effects of ActRU a--e on red blood cell levels are detectable when the compound is dosed at intervals and amounts sufficient to achieve serum concentrations of about 100 ng/ml or greater, for a period of at least about 20 to 30 days Dosing to obtain serum levels of 200 ngml, 500 nginl 1000 ng/ml or greater for a period of at least 20 to 30 days may also be used Bone Mets can be observed at serum levels of about 200 ng/ml, with substantial effects begirming at about 1000 5 ng/ml or higher over a period of at least about 20 to 30 days, Thus, if it is desirable to achieve effects on red blood cells while having little effect on bone, a dosing scheme may be designed to deliver a serum concentration of between about 1.00 and 100 n/ml over a period of about 20 to 30 days. In humans, serum levels of 200 ng/ni may be achieved with a single dose of O mg/kg or greater and semum levels of 1000 ng/nil may be achieved with a 0 single dose of 03 mg/kg or greater. The observed semm half-life of the molecule is between about 20 and 30 days, substantially longer than most c fusion proteins, and thus a sustained effective serum level may he achieved for example, by dosing with about 0.05 to 0.5 T/kg on a weekly or biweekly basis, or higher doses may be used with longer intervals between dosings. For exampledoses orf 1 to I mg/kg might be used on a monthly or bimonthly 25 basis, In certain embodiments, the present invention also provides gene therapy for the in vivo production of ActR I polypeptides. Such therapy would achieve its therapeutic effect by introduction of the Actlla or ActRlb polynucleotide sequences into cells or tissues having the disorders as listed above, Delivery of ActRIl polynucleotide sequences can be achieved 30 using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system Preferred for therapeutic olynucleotide sequences is the use of targeted liposomes Various viral vectors which can be utilized for gene therapy as taught herein indlde adenovirus, herpes virus, vaccinia, or an RNA virus such as a retrovirus. The retroviral 5 vector may be a derivative of a murinc or avian retrovirus Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLVV Harvey murine sarcoma virus (HaMuS .murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV), A number of additional retroviral vectors can incrporate multiple genes. All of these vectors can transfer or incorporate a 0 gene for a selectable marker so that transduced cells can be identified and generated, Retroviral vectors can be made target-specific by attaching. for example, a sugar, a glycolipid, or a protein, Preferred targ'Ig 'is accomupshed by using an antibody. Those of skill in the art will recognize that specific polynucleotide sequences can be inserted into the retriviral genome or attached to a viral envelope to allow target specific delivery of the 5 retroviral vector containing the ActRlH polynuciotidet Altematively, tissue culture cells can be diredly transfected with plasmids encoding the retroviral structural genes gag, po 1 and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest, The resulting cells release the retroviral vector into the culture medium. o Another targeted delivery system for ActRlI polynucleotides is a colloidal dispersion system. Colloidal dispersion systems incle nracromolecule complexes, nanocapsules, microspheres, beads, and lipidbased systems including ci bin-water emulsions, ilicelles mixed micelles, and lipDsomes. The preferred colloidal system of this invention is a Iiposome. iposomes are artificial niembrane vesicles which are useful as delivery vehicles 25 in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active forming (see e g Fraley, et al Trends Bi ocm, Sci., 6:77, 1) Nethods for efficient e transfer using a liposomevebicle, are known in the art see e g, Mannino, et al, Bioteehniques6, 6682 1988. The composition of the liposomne is usually a combination of phospholipids, usually in combination with steroids. 30 especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of' liposomes depend on pIn ionic strength, and the presence of divalent nations, Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphati dyl glycerol Iphosphatidytcholue, phosphatidylserine, 5 phosptid ethanolame, sphiicbroides and gangliosides. Illustrative phospholipids include egg phosphatidytcholinedipalmitoylphosphatidylcholine, and distearoylphosphatidyicholine The targeting of liposomes is also possible based on, for example. organspecificity, cell-specificity, and organel-specificity and is known in the art 10 EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and enbodinents of the present invention, and are not intended to i the invention. IS ExampieIAtila-Fc IeFs Pts Applicants constructed a soluble ActRlla fUsion protein that has the extracellular domain of human ActRila fused to a human or mouse Fc domain with a minimal linker in between. The constructs are referred to as ActRIla-hF and ActRIa-mFc, respectivey. ActRI ahFe is shown below as purified from C-10 cell ines (SEQ ID NO: 7}: 20 ILORSETQECLF FNANW EKDRTNQTG71(5VEPCYGDKDKRRHCFATWKN ISGSIEIVK.QG CWLDDINCY DRTDCVEKKDSPEVYFCCCEGNMCN EKISYFIPEME VTQPTSN PVTPK PPTCGGTI-iH TP PCPAPEL LGGPSVFLFPPKP>KDT LMISRTP EVTCVYVDVSH1 EDPEVKP NW VDGV B :IN A KTKPREEQX NSTY RVVSVLITVLI-HQDW LNGKEYKCKVSN KALP VPtE I 1SL§K9GPBPPQ VYPP SRKEfiIKNOVS'LTCLVKGFYPSD2IA VEWESNQP 25 EN NYKTTPPVLDSDGS FFLYS KLTVDKSRW OGNVFSCSVMIlEA LHNHY TQKAIL S PG-K The ActRIlailhFc and ActRI la-miFe proteins were expressed in CR0 cell lines. Three different leader sequences were considered: (u) Honey bee nellitin (H- BML): MKFLVNVA LVFMVVYlSYIYA (SEQ ID NO: 8) ii Tisue Plasminogen Acti vator (TPA), N.DAMKRLC LuOOV VS ( E (iii) Native: NIOAAA- ;, KLAFAVPLISCSSOiA (SEQ ID NO: 10). The slectd fom emloys he ''PA eade and has the following unpmcessed aino Thaci seuecte omemly NIDAM KRO (;LCCVI.;IsLCCVPVSALOSETELNAWEKTQTVPY 0 D*KDKRRHlCFAT WKNISO("SI "IVK.QCOW - ,, LDDIN CYDRTDOVF EKKDSPP.V YPCCOE DT'LMIS RTI EVTOV ""VVD VSI-I POPE VKF. W) \.D V ' CV PV N AkKI'KER EQYNSTYRVVS 10 VT UOWNKYKKSK P IEISKKQRPVYhPRPMR QVS LIC L -.- 1VKO PYP S PtA VPWPSNC:QPENNYKTTPl-IPV LOt1SOOGSE ELYSKL'Tv 01(RWQ QGCNV FS05 VM\,H EALU.[-1NH-YTQKS LSLSIPCK SEQ I NO,13) This polypeptide is encoded by the fllow ing nucleicaidsune ATOCATOA AGA A.AGOOIOTOTCTOGTOFGCTCTGTOV&AOAC3TCI TITTAATO'C'TAA.TTGOO;AAAAAAGAOACAATOGTAOOGT ATOOTOAC:AA~iATAAC0GC0CATV'F'(,i0CT IAC(T((z'ACT'CATl(>IGG T7TCCATTGCsATAOTC"I'AAACAA(00'I"*TGC; -TGTAAOAATOTT.O CCCATTCAATCAO'TACCCTAOOC CC ACOCTOOTOA,. A~CACACAT CC A A AACCCAAOOACAkC CCATOAT'1C'CCCCYO AOCCI'*"] 0 -A(OOGTO AcwATCG~T GTOO','""(A(-CTOACCACO( A.AO:ACOCTO GAGCTICAAOTTC-.AA("CGOIAC .'^GI00AC 25 OTGAOCAATCACiAAAOOGOAACCATAAAA CACO~'ACOTOTOOCA:(OTOCTACOCOLICCA COAG GAOT'GCTO ,AATGCC AAGOA0TAO1AAOTGCAAOO -'' -F(CCAA.CAAAU -'CCCTCCC',:\-(kl'C(CO ATOiAG AAA AOCATFCTOCAAAOCAAAOO 0 G(CAOOO COOAO.(.;,- , ,-AACCACAOOi CI.'3".'tl'TACACCCjCOO CCAT['CCOOOAOOf--AOATO AC(OAAO AAOCA Oi(lCA GCCTOA l% COTO(, CTGCAAA 30 0 GOTOAjC A3OAAT 0(1( COG AG AO CGCGA A AC.AAOT,-AC AAG AC'A(COCCTO "I k-N(' 'COTOOTOAC 01000ACGO-sCTCCTTOOC 14.T - 0 ATAGCA AGCTC ACC.GT(IGACAAG AGCAGO'FGOCAGCAGOG GAACCTCTTCTAT (GCTCC&ITGATOCATG AGGCTCTOCACA.ACCACTIACA COCAGA AGAGCCTCTCCCTI ) GTCTCCGGGTAAATGAGAATTC (SEQ ID NO:14) Both ActRIla-&e and ActRiIa-mCe were remarkably amenable to recombinant 5 expression. As shown in figure f, the protein was purified as a single.vel-defmed peak of protein N-termninal sequencing revealed a single sequence of ALGRSTQE (SEQ ID NO: 1 1). Purification could be achieved by a series of column chromatography steps including, for example, three or more of the following, in any order protein A chromatograph Q sepharose chromatography, phenyl sepharose chromatography, size exci sion 0 chromatogran chage chroatography The purfication could be completed with viral filtration and buffer exchange. The ActRila-hc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE, ActRlla-hFc and Act Rfla-n showed a high affinity for ligands, particulanly actiyin 15 A. GDP-I I or Activin A ("ActA) were immobilized on a Biacore CMS chip using standard amine coupling procedure ActRla-hFc and ActRIla-mFe proteins were loaded onto the system, and binding was measured. ActRila-he bound to activin with a dissociation constant (Kr) of x10 2 , and the protein bound to GEN A1 with a Ko of 9. 6 x10 See figure 2 ActRilaine behaved similarly. 20 The ActRila-hFc was very stable in pharmacokinetie studies. Rats were closed with I mg/kg, 3 mg/kg or 10 mg/kg of ActRila-hFc protein and plasma levels of the protein were measured at 24, 48. 72, 144 and 168 hours, In a separate study,ats were dosed at I I/kg 10 mg/kg or 30 mgkg, In rats, ActRilahE had an 1 -14 day serum half lfe and circulating levels of the drug were quite high after two weeks (I I pgml, 110 pg/mI or 304 pg/nll for 25 initial administration of 1 mg/kg, 10 mgkg or 30 mg/kg, respectively.) In eynomnlgus monkeys, the plasma half life was substantially greater than 14 days and circulating levels of the drug were 25 pg/ml, 304 pg/mi or 1440 pg/mI for initial administrations of I mg/kg, 10 mg/kg or 30 mg/kg, respectively ExamreasActslla edrBlood Celvelsjn Non-uan Primates A , . ------------------------.. . . . The study employed four groups of five male and five female cynomolgus monkeys each, with three per sex per group scheduled for termination on Day 29, and t wo per sex per group scheduled ri termination on Day 5, Each animal was administered the vehicle S (Group 1) or ActRlla-Fe at doses of 1, 10 or 30 mg/kg (Groups 2, 3 and 4, respectively) via intravenous (IV) injection on Days 1 8, 15 and 22, The dose volume was maintained at 3 mUkg. Various measures of-ed blood cell levels were assessed two days prior to the first administration and at days 15, 29 and 57 (for the remaining two animals) after the first administration 10 The ActRiia-hF causes statistically significant increases in mean red blood cell parameters (red blood cell count [RBC], hemoglobin [FGB, and hematocrit [Hi]) for males and females, at all dose levels and time points throughout the study with accompanying elevations in absolute and relative reticulocyte counts (ARTC; RTC) See Figures 3 -6. 15 Statistical significance was calculated for each treatment group relative to the mean for the treatment group at baseline, Notably) the increases in red blood cell counts and hemoglobin levels are roughly equivalent in magnitude to effects reported with eryt hropoieth, The onset of these effects is more rapid with ActRfla-Fc than with erythropoietin. 20 Similar results were observed with rats and mice. Exapl 3 cta-_erese Red Blood Cell Levels i Huan atns Examlescitc Increases------------ The ActRlia-hFc fusion protein described in Example I was administered to human patients in a randomized, double-blind, placebo-controlled study that was conducted to 25 evaluate, primary, the safety of the protein in heaty, postmenopausal women, Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of Act~llahFc or placebo (5 active: l placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (NI) and 0.03 to OAimg/kg subcutaneously (SC). All subjects were fA lowed for 120 days In addition to pharmacokinetic (PK) analyses the biologicactivity of ActRIkahFc was also 5- W assessed by measurement of biochemical markers of bone fornianon and resorption, and FSH levels, To look for potential changes, hemoglobin and RBC numbers were examined in detail for all subjects over the course of the study and compared to the baseline levels, Platelet 5 counts were compared over the same time as the controL There were no clinically sign-ficant changes from the baseline values over time for the platelet counts. PK analysis of ActRila-h c displayed a linear profile with dose, and a mean half-life of approximately 25-32 days. The area-under-curve (AUC) for ActRila-hFc was linearly related to dose, and the absorption after SC dosing was essentially complete (see Figures 7 and 8). These data indicate that SC is a desirable approach to dosing because it provides equivalent bioavailability and serum-half life for the drug while avoiding the spike in serum concentrations of drug associated with the first few days of IV dosing (see Figure 8). ActRila-hFe caused a rapid, sustained dose-dependent increase in serum levels of bone specific alkaline phosphatase (BAP), which is a marker for anaboo bone growth, and a dose 5 dependent decrease in C-terminal type I collagen telopeptide and tartrate-resistant acid phosphatase 5b levels, which are markers for bone resorption, Other markers, such as PI N P showed inconclusive resul ts. BA P levels showed near saturating effects at the highest dosage of drug, indicating that half-maximal effects on this anabolic bone biomarker could be achieved at a dosage of 0.3 rng/kg, with increases ranging up to 3 mg/kg, Calculated as a 0 relationship of pharmacodynamic effect to AUC for drug, the EC50 is 51,465 (day-ng/ml), See Figure 9, These bone biomarker changes were sustained 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, Overall, there was a very small non-drug related reduction hemoglobin over the 25 first week of the study probably related to study phlebotomy in the 0.01 and 0,03 mg/kg groups whether given IV or SC The 0,i mg/kg SC and IV hemoglobin results were stable or showed modest increases by Day 815 At the 0.3 mg/kg IV dose level there was a clear increase in H1G3 levels seen as early as Day 2 and often peaking at Day 1 5-29 that was not seen in the placebo subjects. At this point in the study, this change has not reached statistical 30 significanCe Overall ActRa-hFe showed a dose-dependent effect on red blood ceil counts and reticulocyte counts. For a summary of hematoiogcal changes, see Figures 10-13, Exanmnle 4: Alternative ActRila~E Proteins A variety of ActRila variants that may be used according to the methods described herein are described in the international Patent Application published as W02006/012627 (see eig, pp. -58) incorporated herein by reference in its entirety An alternative construct ma have a deletion of the C-terminal tail (the fnnal 1 amino acids ofte extracellular domain of ActRIIa, The sequence for such a construct is presented below (Fe portion underined)(SEQ ID NO: 12): I LORSETQECLFFNAN WEKDR TNQTGVFPCYGDKDKRRICFATWKNS$13EIV KQC CWLDD [CYDRTDCVEFKKDSP EVY FCC CEGNMONEKF SYFP EMTCOGCTFPPCPA P ELLOPS Y LF PPKPKDTiISRTPEVTVVVDVSHEDPE K1FN.W D EVNAK TKP RFEOYNSTYRVVSVTV 10D LNKE _ E KALY PIEKFSKA KCOPRE PP EGSKTTPPVLDSDG SFFLYSK5JlrVDKSWOOQNVFlsSVMH EA LHNHYTTOKSLSPCEK Example 5: ActR lb-PFusion Prteins~ Applicants constructed a soluble ActRilb ftsion protein that has the extracellular domain of human ActRilb fused to a human Fe domain A co-crystal structure of Activin and extracelular ActRUb did not show any role for the final (C-terminal) 15 amino acids (referred to as the "tai" herein) of the extracefular domain in ligand binding. This sequence failed to resolve on the crystal structuresuggesting that these residues are present in a flexible loop that did not pack uniformly in the crystal Thompson et al EMBO J. 2003 Apr 5 1 22(7):1555-66. This sequence is also poorly conserved between ActRIlb and ActRita. Accordingly, these residues were omitted in the basic, or background, ActR!b-Fc fusion construct. Additionally, position 64 in the backgrourwd form is occupied by an alanine, which is general considered the "wild type fbrm, although a A64R allele occurs naturally, Thus. the background ActRlbFc fusion has the sequence (F portion underined)(SEQ ID NO:20): SGRG EAETRECY YNANWE LERTNQSG LER CEGEQDKR LCY ASWANSSCTIELVK KCCWLDDFNCYDRQEBC VATE F: NPQ VY FCCCECNFCN ERFTHLPEB ACCTI' PCP('P APE LLEiG PSV.FLFPPKPKDTIL ISRTPE VTCVVVDVSI EDPEVKFNWYV'J EHNA KTKPREEQYNSTYRVVSVLTVI HODQ]WhNGKEY KCKVSNKALP2VPIBKTISk AKGQP 5 REPQVYTLPP-SREEMTIKNQS{ILQCLVKGFPSDIVEWESNQPENNYKTTPPVLDS DSFLYSK VDKSRWOGN YV LSCS VME LHN H YTKSLSLSPGK Surprisingly, the C-terminal tail was fiund to enhance active and GDF- I binding, thus a preferred version of ActRib-Tc has a sequence (Fc portion underine d)(SEQ I) NO:2l)V o SGRGEAEFTRECIYYN AN WE~hRTNQSCLERCECEQDKR LHCYAS WANSSCTIELV K KG CWL DDFNCYDRQEC VATEEiNPQVYFCCCEGNFCNBETH-LPFEAGGP EVTY EPPP I APTCGGfT- PPCPA PEL LOGPSYFLPPKPKDTLMISRTPEVTCVVV) VSH EDP EN' KEN WYVDGVEVHJNA KTKPREEQYNSTIYRVV SVLTVLHODWIVLNGKEYKCKV SNKA LPV- IK T!SKKGQEP R PQVYIPPSRZEEMXKNVSLTC.VKGFYPSDIAVF EESNG 5 OPENNYKPPVLDSDGSFEYSKLTVDKS RWQQGGNVFSCSVMHEALHNHY QS SLSPCGK A variety of ActRllb a variants that may be used according to the methods described herein are described in the inernationa1Patent Application published as W02006/012627 (see e,g., pp, 5960). incorporated herein by reference in its entirety 0 Exampe 6: A CtRib hFc Stimulates Erythropoiesis in Non <Human Primates AciRHb-hEc OgG 1) was administered once a week for Imonth to male and female cynomoigus monkeys by subcutaneous injectior. Forty-eight cynomolgus monkeys (24/sex) were assigned to one ofour treatment groups (6 animals/sex/group) and were administered 25 subcutaneous iqiections of either vehicle or Act~ib-hfc at 3, 10, or 30 mg/kg once weekly for 4 weeks (total of 5 doses). Parameters evaluated included general clinical pathology (hematology, inical chemistry, coagulaion, md urinalysis) AkctRilb-hFc caused statisnically significant elevated mean absolute reticulocyte values by day 15 m treated animalsS By day 36, ActRilb-hFc caused several hemnatological changes, including elevated 30 mean absolute retculocyte and red blood cell distribution width values and lower mean corpuscular hemoglobin concentraton.All treated groups and both sexes were affected.

These effects are consistent with a positive effect of ActRlbAhc on the release of immature reticulocytes from the bone marrow This effect was reversed afer drog was washed out of the treated animals (by study day 56). Accordingly, we conclude that ActRIbahe stimulates erythropoiesis. INCORPORATION BY REFERENCE All publications and patents mentioned heei r erby incorporated by rfrnei their entirety as if each individual publication or patent was specifically and individual indicated to be incorporated by reference. While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive Many variations will become apparent to those skilled in the art upon review of this specification and the elairns below. The full scope of the invention should be determined by reference to the caims, along with their full scope of 5 equivalents, and the spe"iation, along with such variations,

Claims (9)

1. 2. The method of claim I * wherein the polypeptide has one or more of the following characteristics: i) binds to an ActRi ligand with a K of at least 14; and 20 ii) inhibits ActRil signaling in a cell. 3 The method of claim I wherein said poiypeptide is a fusion prot ei including in addition to an ActREl polypeptide domain, one or more polypeptide portions that enhance one or more of in vivo stability; in vivo half life, uptake/administration, tissue localization or di stribution, formation of protein complexes, and/or puification, 25 4 The method of claim 1, wherein said fusion protein includes a polypeptide portion selected from the group consisting of! an imnmunoglobulin Fe domain and a serum albumin. 5, The method of claim I, wherein said polypeptide includes one or more modified anino acid residues selected from: a glycosylated amino acid a PEOvyated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinyated amino acid, an amino acid conjugated to a lipid mniety and an amino acid conjugated to an 5 organic derivatizing agent
2. 6. The method of claim 1. wherein the increase in red blood cell levels is measured as an increase in hemoglobin levels in the blood. 2 A method for treating an anemia, the method comprising administering, to a subject in need thereof an effective amount of an activinu-ActRl antagonist, 10 8. The method of claim 7, whereinthe activinActRil antagonist is an activin or ActRiR antagonist polypeptide,
3. 9. The method of clain 4, wherein the activin-IActRi antagonist polypeptide is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO2; b) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:3; and C) a polypeptide comprising at least 50 consecutive amino acids selected from SEQ ID NO: 2 20 d) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:16; e a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO 17; and f)a polypeptide comprising at least -0 consecutive amino acids selected from SEQ ID 25 NO: 16.,
4. 10. The method ofclaim 9, wherein the activin-ActRll antagonist polypeptide has one or more of the following characteristics: i) binds to an ActR I I igand with a Kl: ofat least l04 M; and ii) inhibits AetRil signaling ina cell, I I The method of claim 9 wherein said activinArtR iN antagonist polypeptide is a fusion protein including, in addition to said activin-ActRl1 antagonist polypeptide, one or more polypeptide portions that enhance one or more of in vivo stabintyn vivo half 5 ife uptakeadministration, issue localization or distribution, formation of protein complexes, and/or purificationt
5. 12. The method of claim 11, wherein said fusion protein includes a polypeptide portion selected from the gwup consisting of an immunoglobulin domin and a serumn ahlbunin 10 1 The method of claim 8, wherein said activin or ActRUi antagonist polypeptide includes one or more modified amino acid residues selected from a glycosylated amino acida PEGylated amino acid, a famoesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatzing agent. 15 14, A method of identifying an agent that increases red blood cell levels, the method comprising a) identifying a test agent thit binds to a ligandbinding domain of an ActRil polypeptide competitively with an ActRII ligand; and b) evaluating the effect of the agent on red blood cell levels in an animal. 20 15. Use of an activin or ActRil antagonist polypeptide for making a medicamentfor increasing red blood cell levels in a human patient, 16, A method for increasing red blood cell levels in a human patient, the method comprising administering to the patient an effeive amount of an ActRI-Fe fusion protein, wherein the ActR I lFc fusion protein comprises an amino acid sequence 25 selected from the group consisting oft a) an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQt ID NO3, b) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID N03, c) the amino acid sequence of SEQ ID NO:3, d) the amino acid sequence of SEQ ID NO:2, e) the anino acid sequence of SEQ ID N O-7, f) an amino acid sequence that is at least 90% identical to the arino acid sequence of 5 SEQ ID NO:17, g) an amino acid sequence that is at least 95% identi cai to the amino acid sequence of SEQ IDNO:17, h) the amino acid sequence of SEQ ID NO- 17, i) the amino acid sequence of SEQ ID NO: 16, 10 j) the amio acid sequence of SEQ ID NO:20, and k) the amino acid sequence of SEQ ID NO:2 1, 17, The method of claim 16, wherein the method causes less than 15% increase in the patient s skeletal muscle mass,
6. 18. The method of claim 16. wherein the ActRi-Ec fusion protein is administered so as to 15 reach a serum concentration in the patient of at least 100 ng/m for a period of about 20 to 30 days.
7. 19. The method of claim 16 wherein the ActRilFe fusion protein is administered so as to reach a serum concentration in the patient in the range of 100 ng/ml to 1000 ng/nm
8. 20. The method of claim 16, wherein the ActRIl-Fe fusion protein has a serum half-life of 20 between I5 and 30 days, 21 The method of claim 20, wherein the AetRii-Fe fusion protein is administered to the patient no more frequently than once per week
9. 22. The iethod of claim 20, wherein the ActRIl-Fe fusion protein is administered to the patient no more frequently than once per month. - 60 -