AU2013221910B2 - 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 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

The mature red blood cell, or erythrocyte, is responsible for oxygen transport in the circulator}' 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 (pOi) and delivers oxygen to areas of the body with a relatively low pCE-

Mature red blood cells are produced from pluripotent hematopoietic stem cells in a 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 ceil mass in the body, and production may increase or decrease in response to various stimuli, including 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 rciiculocytcicrythrocytc 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 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 erythropoiesis primarily in the spleen.

Various forms of recombinant 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 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 (Weatherall & Provan (2000) Lancet 355, 1169-1175). Anemia may result from a reduced rate of production or increased rate of destruction of red blood cells or by loss of red blood cells due to bleeding. Anemia may result from a variety of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation.

Treatment with Epo typically causes a rise in hemoglobins by about 1-3 g/dL in healthy humans over a period of weeks. When administered to anemic individuals, this treatment regimen often 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 many individuals are refractory to even high doses (Horl et al. (2000) Nephrol Dial Transplant 15, 43-50). Over 50% of patients with cancer have an inadequate response to Epo, approximately 10% with end-stage renal disease are hyporesponsive (Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998) J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplastic syndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67). Several factors, including inflammation, iron and vitamin deficiency, inadequate dialysis, aluminum toxicity, and hyperparathyroidism may predict a poor therapeutic response, the molecular mechanisms of resistance to Epo are as yet unclear.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

In one embodiment, the present invention relates to alternative compositions and methods for increasing red blood cell levels in patients.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a method for promoting erythropoiesis in a patient, the method comprising administering to a patient in need thereof an effective amount of an ActRII polypeptide selected from: a) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2; b) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:3; c) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; d) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17; e) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:7; f) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 12; g) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:20; and h) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:21.

According to a second aspect, the present invention provides use of an ActRII polypeptide selected from: a) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2; b) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:3; c) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; d) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17; e) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:7; f) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 12; g) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:20; and h) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:21. for the manufacture of a medicament for promoting erythropoiesis.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

In part, the disclosure demonstrates that activin antagonists, as well as ActRIIa and ActRIIb antagonists, can be used to increase red blood cell and hemoglobin levels. In particular, the disclosure demonstrates that a soluble form of ActRIIa acts as an inhibitor of activin and, when administered in vivo, increases red blood cell levels in the blood. A milder effect was observed with a soluble form of ActRIIb, which binds Activin A with lesser affinity than soluble ActRIIa. While soluble ActRIIa and ActRIIb may affect red blood cell levels through a mechanism other than activin antagonism, the disclosure nonetheless demonstrates that desirable therapeutic agents may be selected on the basis of activin antagonism or ActRII antagonism or both. Such agents are referred to collectively as activin-ActRII antagonists. Therefore, in certain embodiments, the disclosure provides methods for using activin-ActRII antagonists, including, for example, activin-binding ActRIIa polypeptides, activin-binding ActRIIb polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, anti-ActRIIb antibodies, activin-, ActRIIb-, or ActRIIa-targeted small molecules and aptamers, and nucleic acids that decrease expression of activin, ActRIIb, or ActRIIa, 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, activin-ActRIIa antagonists can be used to promote bone growth and increase bone density. As described 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-ActRIIa 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. This selective effect may be achieved by using, for example, lower doses of activin-ActRIIa antagonist, less frequent doses, or by using an activin-ActRIIa antagonist with a shorter serum half-life 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 ActRIIa polypeptide or an ActRIIb polypeptide. ActRII polypeptides may be formulated as a pharmaceutical preparation comprising the activin-binding ActRII polypeptide and a -—5- pharmaceUticMIy acceptable carrier. The aehvimbinding ActRII polypeptide may bind to aefryiu with a Κρ less than t micrornoiar or less than 100,: 10 or! oaoomolar,: Optionally, the acti yin-binding AeiRil polypeptide selectively binds actiyin versos GDFt1 and/or GOBS, and optionally Whir a fro that M dt least 10-ib!d, 20-fold or 50*ibid lower with respect to aehvin than with fespect to GOF11 and/otGDPS. While not wishing to be bound to a particular meehamsm Of action, it is expected that this degree of selectivity for act! vin:inhibition over GDE11 /GORE inhibition accounts for effoets on bene or erythropoiesis withqfit a consistent!y measurable effect on muscle. In many ernbodimenis, an ActRII polypeptide will be selected for causing less than 1.5%, less than 10% or less than 5% increase in fopscle at doses that achieve desirable effects on red Mood cell levels, lire 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 activinfoipding AetRIIa polypeptide for use in such a preparation may be any of those disclosed herein, such as a polypeptide having ah amino acid seduenoe selected from SEQ ID MOs: 2, 3, 7 or 12, or having an amino acid sequence that is at least 10%, 85%, 90%, 95%, 97% or 99% identicaltoan amino acid sequence selected from SEQ ID NOs: 2, 3, 7, M or 13. An activin/binding ActRIia polypeptide may include a functional fragment of a natural ActR!la polypeptide, such as one comprising at least l0,20 or 30 amino acids of a sequence selected from SEQ ID NOs> 1-3 or a sequence of SEQ ID MO: 2, lacking the C-terminal 10 to 15 amino acids (the "Half % An add van-binding AetRIib polypeptide for use in such a: preparation inay he: any of those disclosed herein, such as a polypeptide having an amino add sequence selected from SEQ ID NGs; Id, 17j 20, or 21 or having an amiho acid Sequence that is at least 80%, 85%, 90%, 95%, 97% or 99%: identical to an aminp acid sequence selected from SEQ ID NOs: 16, 17, 20, or 21, An achvinfoinding ActRilb polypeptide may include a functional fragment of a natural ActRIlb polypeptide, such as due comprising at least 10,20 or 30 ammo acids of SEQ ID HQs: 15-17 or a sequence laefeing the C-toummal 10 to 15 amino acids (the ΟηΙΓΤ such as SEQ ID NO: 17. A soluble, achvin-binding ActRII polypeptide may include one or more alterations in the amino acid sequence (e.g., in the ligand-binding domainf relative to a naturally occurring AefEIl polypeptide. Examples of altered Achilla and AeiRilb 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 the polypeptide when produced in a eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRIl polypeptide.

An activin~hindin.g AciRO polypeptide may te a fusion protein that has, as one .domain» an ActE.lI polypeptide, (e;.g., a tigapd-btridipg pdttioii of ad AciRIIa or A.etRllb) and one or more additional domains that provide a desirable property, such as improved pharmacokinetics,; easier purificatiorp targeting to particular tissues, etc. For example, a domain of a id$3.0^^tetn:may'«$th^0'^e or more of in vivo stability, in vivo half life, uptake/admimstration, tissue loealization or distribution, ton-nation of protein complexes, muitimerixation of the fusion protein, and/or puniication. An aeitviofeinding AetRll fusion protein may mehide an immunoglobulin Fo domain (wild-type or mutant) or a serum albumin or other polypeptide portion that provides desifable properties such as improved pharmacokinetics, improved solubility or Improved lability. In a preferred embodiment, an AetRH-Fe fusion comprises a relatively unstructured linker positioned between the Pc domain and the extfaeelluiar AefRll domain. Ibis unstructured linker may correspond ip the roughly IS amino acid ujisfeuctnred region at the CUerminal end of the extracellular domain of AefRilfth® ^1¾ or it may he an artificial sequence of 1, 2, 3,4 or S amino acids or a length pfbetween S and 15.20,30, SO or more ammo adds that are mlativeSy free of secondary sfracture, or a mixture of both. A linker may berieh ip glycine and proline residues and: may, for example, contain a single sequence ofthreonine/serine and glycines or repeating sequences of tlirepnine/serine and glycines (e.g,51¾ or SO# singlets or repeats). A fusion protein may include a purification subsequence, such as an epitope tag, a:FLAG tag, a pchyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIl polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a FEGylated amino acid, a farnesylated amino acid, an acetyiated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid eonjugafed to an organic fieri vatixing agent. A pbarmaeeuti oaf preparation may also include one or more additional compounds such as a compound that is used to treat a bone disorder. Rreferahlv, a pharmaceoticai pfeparaiioTi is substantially pyrogen free. In general:, it is preferable that ah: AetRii protein be expressed inn mammalian ceil line that mediates suitably natural glyeosylation of the AetRii protein so as to diminish the likelihood of an unfavorable immune response in a patient. Human and CEO ceil lines have been used successfully, and fi is expected that other common mammalian expression systems will be useful

As described herein, ActRIIa proteins designated /£!etEH«ί#c^(¾:.f$1rm:wH¾ a minimal hnkefbefween the.ActRIIa portion and IheFc portion^ have desirable pmgerties, including selective binding to adtivin versus GDF8 and/or GDF11, high affinityligand binding and serum half life greater than two weeks in animal models. In certain embodiments the invention povides ActR:II-Fc pol^epbdes and pharmaceutical preparations comprising such polypeptides and a pharmaceutically acceptable excipient

In certain aspects* the disclosure provides nucleic acids encoding a soluble activin-binding ActRii polypeptide, such as an ActRIIa or AetRllb polypeptide. An isolated polynucleotide may comprise a coding sequence lor a soluble, activimbinding AetRil polypeptide, such as described above. For example, an isolated nucleic acid may include a sequencecoding forauMtmeelinlar domain (e.g., ligand-binding domain) of an ActRii and a sequence that would code for part or all of the iranaptcnfemrie domain and/or the cytoplasmic domain of an ActRii, but tor a stop codon positioned within the transmembrane domain or the cytoplasmic domain, dr positioned between the extracellular domain and the transmenshrane domain or cytoplasmic domain. For example, an isbl^i^:pb|ynael#i3bsaay comprise a full~Iengdi ActRIIa polynucleotide sequence such as SEQ ID MO: 4 or 5 or a fell-length ActROb polynucleotide sequence such as SEQ Ip NO: 18, or a partially truncated version of ActRIIa or ActRIIh, said isolated polynucleotide further comprising a transcription termination codon: at least six hundred: nucleotides before the 3?~terminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused: to a truncated portion of a foil-length ActRii. A preferred nucleic acid: sequence for ActRIIa is SEQ ID NO: 14, Nucleic acids disclosed herein may he operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides. Preferably the cell is a mammalian cell such as a OHO cell.

In certain aspects, the disclosure provides methods for making a soluble, activin-hinding ActRII polypeptide. Such a method may include expressing any of the nucleic acids (e.g., SEQ ID NO: 4, 5 14, 18, or 19} disclosed herein in a suitable ceil, such as a Chinese hamster ovary (CHO) cell. Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble ActRii polypeptide, wherein with a soluble ActRii expression construct; and: b) recovering the soluble ActRii polypeptide so expressed. Soluble AetRil polypeptides may berecovered as crude, partially purified or highly purified factions. ; by; .a series of purification steps, including, for example* one, two or three or more of the following. ih any order: protem A chromatography, anion exchange chromatography (e,g.s sepharose|s lydrophqhie interaction chromatography |e.g-> phenylsephapose^-sfee exctosion chrofoatography, arid: cation exchange: chromatography. in certain aspects* an a<foyio-AeiRit antagonist disclosed herein, such as a soluble, activin-binding AetRlIa polypeptide or soluble, activio-binding ActROb polypeptide, may he used in a method for promoting red biopd cel! production or increasing red Mood cell levels in a subject. In certain embodiments, the diseiosure provides methods for treating a disorder associated with low red blood pell counts or low hemoglobin levels (e.g., an anemia), or to promote red blood cell .production, in patients in need thereof A method may comprise administering to a subject ip need thereof an effective amount of activityActRil antagonist, in certain aspects, die disclosure provides uses of acfivin-ActRii antagonists for making a medicament for the treatment of a disorder or condition as described herein.

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 aetivin or a ligand-binding domain of an ActRJI polypeptide; and b) evaieatmg th e effect of the agent on the levels of rod blood cells, hemoglobin, and/or red blood cell precursor levels (e.g., reticulocyte levels).

BRIEF DiSCRin iOM OF THE DRAWINGS

Figure 1 shows the pnriBcation of ActRlla^hFc expressed in CHO cells, The protein purifies as a single, well-defined peak as visualized by sizing column (left panel) and Coomassle stained SDS-PAGE (ri^it panel) (let lane: molecular weight standards; right lane: ActRila-hFc),

Figure 2 shows the binding of ActRlIa-bFc to activinund GDF-Π, as measured by BiaCora!M assay.

Figure d shows the effects of AetRlla-bFc on red blood cell counts in female nonhuman primates. Female eynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 m^kg or 30 mg/kg of AetRlIa-hPe on day 0, day 7^, day \ 4 and day 2 L 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 d shows the effects of ActRiIa~hFc on red blood: cell counts in male non-human primates. Mate, eynomol gus nfonkeys groups of fi ve monkeys each) were treated with placebo or lmg(fe§s10mg/kg or dll mg/kg: of ActRlla-hFc on day 0, day f, day 14 and day 21. Figure 4Λ shews red blood cell (RBC) counts. Figure 48 shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57 s two monkeys remained in each group.

Figure 5 shows the effects of ActRlla-hFc on reticuiocyte counts in female nonhuman primates. Cynomolgus mppkeys (foot groups of five monkeys each) wore treated with placebo or I mg/kg, 10 mg/kg or 30 mp'kg of AeiRIla-hFc on day 0« day 7. day 14 and day 2 L Figure 5A shows absolute reticulocyte counts. Figure SB shows the percentage of reticulocytes relative to RlBCs, Statistical siptificance is relative to baseline for each group. At day 57, two rnonkeys remained in each group.

Figure 6 shows the effects of AotRlia-hFc on reticulocyte counts in female non* human primates. C^ompigP3,:X»onk^':(ip«r groups of five monkeys each) were treated with placebo or 1 mg/kg; 10 mg/kg or 3 Θ mg/kg of AetElla-hlo on day 0, day 7,}: day 14 attd; day 21.. Figure 6 A shows absOlnfoiretieulocfte counts. Figure 68 shows the pereentagg of reticulocytes relative: to RBCs, Statistical slgrTificance is relative to baseline for each group. At day 57, two monkeys remained in each group.

Figure 7 shows results from the human:clinical tfial described in Example 5, where the areamhder-curve (AUC) and administered dose of ActRlla-hFc have a linear correlation, regardless of whether ActRlla-hFc Was athnirn stored intravenously (IV) or subcutaneously (SC),

Figure 8 shows a comparison of serum levels of ActRlla-hFc in patients administered IV or SC.

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

Figure Ifi depictsthe median change fibm baseline of hematocrit levels -from the human clinical trial described in Example 5. AeiRIia-hFo Was administered intravenously (IV): at the indicated dosage.

Figure; 1 i depicts' Ihe median change from baseline of hemoglobin levels Rom; the human clinical trial described in Example 5, AeiRlIa-hFe Was administered mfravendpely (IV) at the indicated dosage. figure 12 depicts the medianchange Rom Baseline of RBC (red blood ceil) count from the human clinical trial described in Example S. AetRila-hfc was administered intravenously (IV) at the Indicated dosage.

Figure 13 depicts the median changefrom baseline of^rctlculpcyte count h»m the human clinical trial described in Example 5. ActRIla-hFe was admimstered intravenously (IV) at the Indicated dosage.

DETAILED DESCRIPTION OF THE INVENTION 1- Overview

The transforming gtx>wlb fectordscta ;(TC3F-beta) soperikrvily contains di variety of growth factors that share: common sequence elements and structural motifs. These proteins are known to exert Biological effects on a large variety of cell types in Both vertebrates and invertebrates. Members of the snperfamily perform important functions during embryonic development in pattern formation and tissue specification andcan influence a variety of differentiation processes* including adipogcoesiss myogenesis, ehondrogeoesis* cardiOgenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two genera! Branches: the BMP/GDF and the TOF-beta/ActivimBMElf) branches* whose memBers have diverse* oflen complementary efifeets. By manipulating the activity of a member of the TGF-Beta family* it is often possible to eause significant physiotogieal changes in an organism. For example, the Piedmontese and Belgian Bine cattle Breeds carry a loss-Of-function mutation .intheODF0v(aIso called myostatin) gene that causes

Grobet et aL* Nat Genet, 1997, 170 ):71-4. Furthermore, in humans, inactive alleles oFQDFI are assodated with increased musde mass and* reportedly, exceptional strength, ScBudtke et al, N Eogl i Med 2004, 350.2682-8.

Aetivins am dimeric polypeptide growth factors that belong to the TGF-heta superfamily. There are three principal activin forms (A. B, and ABj that arc homo/heierodimers of two closely related p sufeunits (PaPas βεΡδ^ and βΑββ, respectively).

The human genome also encodes an activin C and an activin £, which are primarily expressed in the li^*..aad;feelct®diineric forms containing pc or βε are also known. In the TGF-beta superffenby, dcrivins are unique and moltifonctionai fetors $iat can stimulate hormone production in ovarian and placental pells» support neuronal cel! survival, inSnence cell-cycle progress positively or negatively dependmgon cel! type, and induce mesodermal differentiation at least in amphibian emhryoa{0eFadlo:0t aL, 1991, Free Soc Ip Bio! Med. 198:500-112' Dyson ef al, 1997, Curr Biot 7:81-84; Woodruff 1998, Btoehem IfoarmacoL 55:953-983;)- Moreover, erythrold differentiation:factor (EDF) isolated fern the stimulated human monocytic leukemic cells was found to be; identical to activin Λ (Murats et al., 1988, FNASi 85:2434). It has been suggested that activin-A promotes erythfopoicsis in the bone marrow,: In several tissues,: activin signaling is antagonized by its related heterodimer, inbibin. For example, dupng the release offolEele-stimulatthg'hormone (FSH) from the pituitary, activin promotes FSif secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that may regulate activin hsoactivity apd/br bind to activin include follistatin (FS|, foHistatm-relaied protein (FSRP) and as-macroglobulim TGP-fi signals are mediated by heteromerie complexes of type 1 and type 11 serine/ threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massague, 2000, Flat: Rev, Mol, Cel! Biol, 1:169-178), These type I and type 11 receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with eysteine-nch region, atransmembrane domain, and a cytoplasmic domain with .pr^Cfed :^ae/thtpon|b%^eeISeity» Type 1 receptors are essential for signaling; and type 11 receptors are required for binding ligands and for expression of type 1 receptors. Type 1 and 11 activin receptors form a stable complex aherhgaud binding, resulting in phosphorylation of type I receptors by type II receptors.

Two related type II receptors (AetRli), ActfeHa and AetRllb, have been identified as the type II receptors for aetivins (Mathews and Vale. 1991, Cell 65:973-982; Atiisano et sl,f 1992, Cell 68: 97-1:08), Besides aetivins, AcfRIIa and ActRHb can bipchemicaily interact ^ with several other TGF-β family proteins, including BMP?, Nodal GDF8, and GDFI1 (Yamashita et al,, 1995, i. Cell Biol. 13:0:217-226; Lee and McPherron, 2001, Proc, Natl, Acad. Sci. 98:9306-9311; Yee and Whitman, 200 i» Mol, Gel! 1:: 949-957;: ©h et a!,, 2002,

Genes Dev, 10:2749-54). AftKA is the primary type I receptor foraetivins, particularly for activin A, and ALK-7 may serve as a receptor for aetivins as well, particularly for activin B,

As desnoiBtrated herein^ a sohfole AetStlja polypeptide (sAclRlla), which shows substantial greforehee ip feindrogto activin A as opposed to other ITSF-beta innily members, such as GPfS or C3DF! 1, is effective to iporease red blood cell levels in vivo. Whilenot wishing to ho bound to any particniar mechanism, it is expected that the effect of sAetRHa is caused pritparily by an activm antagonist effect, given the very strong activin binding (picornolar dissociation constantl exhibited by the particular sAcfRila construct tised in these Studies. Regardless of mechanism, it is apparent Irom this diselosure that AetRiia-aetivin antagonists increase red blood ceil levels in rodents, monkeys and humans. it should be noted that hematopoiesis is a complex processs regulated fey a variety of factors, including erythropoietin, G~QW -and The terms ^increase red blood eeh levels” and 4'promote red blood cell formation^ refer to clinically observable metrics, such as hematocrit, red blood cell counts and bcmoglobin measureinents, and are intended to be neutral as to the mechanisrh by which such changes occur.

As also demonstrated herein, a soluble AetRllb polypeptide (sActRllb) is effectiveto increase reticulocyte levels in vivo, an effect which, over a longer time period is expected to cause increased hemotocrit levels.

The data reported herein with respect to non-human primates are reproducible in mice* tots and humans as well, and therefore, this disclosure provides methods for using AeiRll polypeptides and other activin-ApfftH antagopists fo promote red blood cell production and increase red blood cell levels in mammals ranging from rodents to .-humans. Activin-ActRIl antagonists include, for example, a;ptiyijrirbindi»g sol»bfe'Ai^R:|lili polypeptides, aclivin-binding soluble AetRllb polypeptides, antibodies that bind to activin {particularly the activin A or B subunits, also referred to as β.Α or βΒ) and disrupt ActRlla tmeifor AetRllb binding, antibodies that bind to ActRlla and disrupt activin binding, antibodies that bind to AetRllb and disrupt activin binding, non-antibody proteins selected for activin, AetRfib or ActRlla binding ($¢0 0.1., WO/2O02/088171, WO/2006/055689, and WO/2002/032925 for examples of such proteins and methods for design and selection of sajne}, random:i2ed :peptides selected for activin, AciRUb,:0r AeiEllahihding, often affixed to an Fe domain, Tvfo different proteins for Other moieties} with activin, AetRllb, or ActRlla binding activity, especially activin binders that block the type 1 (e.g., a soluble: type 1 activin receptor) and type II (e.g., a soluble type 11 aetivih receptor):'binding sites, respectively, may be linked together to create a hi&amp;netional binding molecule, Mitelelc acid aptamers, small molecules and other agents that inhibit the aetivm-AciRII signaling: axis are included as aelivm-AeiRIl antagonists. Various proteins have aetivin-ActRIi anmgphist activity, including inhihin (fce., inhihin alpha subunit), although inMbin does not umversally antagonize activin in all tissues, fellsstatin {e.g., fol1istatin-28S and ibUistatin-S 15), FSRP, activin C, alpha{2)maaerogjobulin5 and an Ml08A (methionine to alanine change at position i OS) mutant activin A, Oencrally, alternative forms of activin, particularly those with alterations in the type I receptor binding domain can bind to type II receptors and tail to form an active ternary complex; thus acting as antagonists. Additionally, nucleic acids, such as antisense molecules, siRls As or rihozymes that inhibit activin A, B, C or E, or, particularly, AeiRIIaor ActRllh expression, can be used as aetivin-ActRIi antagonists. The activin-AetRl! antagonist to be used may exhibit selectivity for inhibiting activin-mediated signaiing versus other members of the TQF-beta family, and particularly with respect to 013F§ and :G;0FT|:, lie tenns used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the speeihe context where each term is used. Certain terms are discussed below or elsewhere lh the specification, to provide additional guidance to: the practitioner in describing the compositions and methods of foe invention: and liow to make andnse them. The scope or meaning of any use of a term will be apparent1 from the specific context in which the term is used. “About’'* and “approximately” shall generally mean an acceptable degree of error for the i|uantity measnfecl given: the nature or precision of the measurements, Typically, exemplary degrees of error are: whhin 20 percent (%}, preferably within 10%,; and more preferably within 5% of a given value or range of values,

Alternatively, and particularly in biological system^ 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 of a gi ven value; Numerical quantifies given herein are approximate unless stated otherwise^ meaning that the term “about” or 'Approximately” can he inferred when: not expressly stated. may induce steps of ceftnpadng sequences to each other, Inducing wildrtype sequence to one or more mutants (sequence variants), Such comparisons typically comprise alignmMts of polymer sequences, e,g., using sequence alignment programs andfer algorifetns that are well known in tire ait (for example, BLAST, PASTA ant! MEGAiTGH, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation cohfams a residue insertion or deletion, the sequence alignment will introduce a “gap* (typically represented by a dash, or *%”) in fee polymer sequence not containing the inserted or deleted residue. “Homologous/' in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,“ including pmfeinsfrom superfemilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic aci ds) have sequence homology, as rofleeted by their sequence similarity, whether in terms of percent identity or by the pesenee of specidc residues or motifs and conserved positions.

The term “sequence in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino aoid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the tend “homologous/' when modified with an adverb such as “highly/1 may refer to sequence similarity add: may or may not relate to a common evolutionary origin. %. ActRlI Polypeptides

In certain aspects, the present in vendorr relates to AetRli polypeptides. As used herein, the term "Aetfeir’ refers to the family of type 1! aetivin receptors, This family includes both the aetivin receptor type 11a and the aetivin receptor type iih. lb certain aspects, the present invention relates to ActR.Ua polypeptides. As used herein, the derm “ActlHla1' refers to a family of aetivin receptor type Ua /AetRlla) proteins from any species and variants derived from such ActRIIs proteins by mutagenesis or other modification.: Reference to AetRlla herein is understood to be a reference to any one of the currently identified forms, Members of the AetRlla family are generally transmembraue proteins, composed pf a liganddfonding extracellular domainwith a cysteine^ich region, a tmnsmembraneidomam,, and a cytoplasmic domain with predicted serine/forcanine kinase activity.

The term "AetR lla polypeptide” includes polypeptides comprising any naturally occurring polypeptide of an ActRlla family member as well as any variants thereof (including m:foant% Augments, fhsipns, and peptidomimetic forms} that retain a useful activity. See, for example, WQ/lOid/012627, Por example, ActRlla polypeptides include polypeptides deri ved Rom foe sequence of any known ActRlla haying a sequence at least about 80% identMi to the seguenee of an ActRlla polypeptide, and optionally at least 85%, 9ϋ%, $5%ν 97%:ϊ f9% or great er identi ty , For example, an ActRlfo polypeptide of the Invention niay bind to: and inhibit the function of an ActRlla. protein and/or aetiym. An ActRlla polypeptide may foe selected for activity in promoting red bipod cell formation in vivo. Examples of ActRlla polypeptides includolntman ActRlia precursor polypeptide (SEQ ID NO: I) and soluble human ActRlla polypeptides (C-g., SEQ ID Ndst 2,3, 7 and 17},

The human ActRlla precursor protein sequence fe as follows: M LIAS IVIC A 5% V Y EH H Khi A YP f VL V PTQ DPQFPPPSP HG L K P ί .,01 HE VKARGRFi3CV¥RAQLfo^EyyAyKlFPXQDRQS^QHEyE¥:ySLPGHKHEh X LQ Γ X EAEREGTS V DRRLWL X TA FH E KGS LS &amp;RLKANWS WEE EC Η X AE FMARCSLSYLHEDIPGBRDGERPAISHRDlKSKMELLKRNLFfoGXADFGL ALKFSAGKS AG DI BGQVGT RE YRIAP E VLE S AI If FQE 0&amp;FX.R I DMYARGX. VLmLASRCTAADGPVOEYMLPFBEEXGQH PSX»E DMdSVWBKRKREtfL EDYWQKHAGEAMLC ETI EEGW D fXDASARL S AGE VGERl Ι0Μζ>Εί ·Τ:Ν IIT TEDXW:¥¥TRVTR¥OFPPKS:SSL (SEQ ID NO: !}

The signal peptide is single underlined; the extracellular domain :s in hold and the potential Nfoinked glyeosylatfon sites are double underlined.

The human AelRIIa soluble (extraedlukrh processed polypeptide sequence is as follows: TOGAS ETQEGLFFMA NWEE DRT M QTCSVE PC Y G D K DKRRHCEAT W ΕΝ IS G SI Ei I V:FCQSC p;L Dpi S C f ORT DC VΕΚ K PS E Sy Y FCC C ESWMCN E K FS Y FF EMEVTQPTSNPYTPKPP (SEQ IDNOxY)

The C-teminal^taiT5 of the extraceihtl&amp;r domain is underlined, The sequence with the “tail” delated (a A15, sequence) Is as follows: XLGRSETQECLFF^ANMEKDRTISIQTGVEFCYGOKDKEKHCF.ATWKMISG SIS1 VKQGCWL D DISC Y DRTDCVEKKOSPEV Y FCCC BGNMCWS K FSYFP EH (SEQ ID NO:3}

Tite niiclcie acid sequence eueodlng iTOman AetRlIa precursor protein is as follows (nucleotides 164-1 70S of GenMnfc entry NM.J)0!61&amp;):

ItTGGGaGCTGeiCCA^aCTTGGCGTTTGCCGTCTTTCTT/vrCTeCTGTT

CTTCAGGTGCTATACTTGGTASATGAGiYRACICAGGAGTGTCTYTITSIT

TMTGCTMTTGGGAaaaaGAGaGaaccaaTcaA^CTGGTGfTGMccG

TGTTATGSfcACAAAGATAAAEGGGGGeATYGTTTTGeTACCTGGAAGA aTaiTTeTGGTTceaTTGh^.TaGTGAaacaaGGTTGTTGGeTGG^TGa

TA?CAACTG€TA?SACAGGA€7:GATTGTSTAGAAAAAAAAGACAG€€GT

GAAGTATATTTTTGTTGCTGTGAGGGGAATATGTGTAATGAAAfoSTTTT CTTSTTTTCCAGAGATGGAAGTCACACAGCCCaCTTCimATCCAGTTAe

AGeTAAGCCACCCmTTftCimGATeCTGGTCTATTCGTTGGTGeGACTT

ATSTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGeATC aCAAGATGGCCTACGCfCCTGTlETTGTTCCaACTCAAGACCCAGGAGC: ACCGCCACCTTCTCCftTTACTAGGGTTGAAaGCaCTGGAGTTATTAGAft

GTGAAAGCAAGGGGAAGATTTGGTTGTGTClGGA&amp;ftGCeCAGTTGCTTA

ACGAATATGT G G CT GTCAAAATAXTTCCA&amp;TACAGGAGAA&amp;CAGT CAT G

GCAftAM’GAATAGGAAGTC'rACAGTTTGCCTGGASTGAAGCATGAGAAC

ATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGAIGTGG

ATG T TTS SC TG AT C ACAG C ATTTC AT GA AAAG GGI T € A CTA7CAG&amp;C T T TGTTAAGGG1TAAXGTGGTCTGTXGGAATGAACTGTGfCATATTGCAGAA

AGCATGGCTAGAGGftTTGGCATATfTACATGAGGATATACCTGGGCTAA aagatggccagaaacctgccatateicacagggacatcaaaagtsaaaa

6CCTi^^tiTG^SGCTG(SCA&amp;G,rC!taCAGSG:SAT^eG:©ATGGftCAGG TTGGWCCQSaGGTAGATiGCfCCAi&amp;GG:T^TTft0a6GGf:C5Cf^T^ GTTGCAa^GGGATGCATTTTTGAGGATAG^GlTGTATGGC^TGGG^TTB GTeGGATGGGimGTGGeT^G'rCGGTGTA.CTGGTGeAGATGGACCI’GTAG ATGAOTGATGTTGGCATTTGAGGlGGAAAGfGGGGAGCATCeATCTCT f G AAGAG ATGGAGG AAGTT GTT ST GGATAAAAAAAAGASGGCTGTT TTA AGAGATTATTGGCAGAAAeATSeTGGAATGGeAATGGTSTSTSAAACCA TTGMGAATGTTGGGATCAGGACGCAGAAGGGAGGTTATeAGGTQGATG TGTASGTGAAAGAAITAGSGAGATGCAGAGACTAACAAAlATTATTACe AGAGAGGACATTGTAACAGTGGTCAGASTGGTGACAAATGTTGAGTTTC CTCCGAAftGAATeGAGTCTMGA {SBQ ID NO: 4}

The nuefeie: acid: sequence encoding a human AciRIIa soluble (extracellular) |xdypepiide is as fellows:.

ATAST TGGTAGATCAGAAACTCASGAGTGTCT TT TGT T fAAIGCTAATT gggasaaagagagaacgaatsaaactggtgttgaagcstgttatggtga CAAAGATAAACGGCGGCATTGTTTTGCTACeTGGAAGAftTATTTCTGGT TC CAT TGAAAT AGTGAAAGAAGGTTGT TGGCTGB ATS ATAT C RACT GCT ATGAaAGGAGTGATTGTGTSSAAAAAAAAGACAGCGCTGAAGTATATTT TT GTT GCTST GAGGGGAATATGTGTAA TGAAAAGTTTT GTTAT T T TGGA GAGATSGAAGTCAG&amp;eAGCCeAGTTCAAATGCAGTTAeACeTAAGCGAC CC (SBQ IB NO: 5} 1« certain aspects^ the present invention relates to AciRIIb polypeptides. As used herein, the term “ActRIIb*’ refers to a fendly ufiaeiivm receptor type lib (ActROb) proteins from any species and variants derived from such AetROb proteins bv mutagenesis or other modification. Reference to AetRIIb herein is understood to be a reference to any one of the cnrremly identified forms, Members of the ActROb femily are generally tmnsmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region., a transmembrane domain, and a cytoplasmic domain with predicted senhe/threonine kinase activity.

The term “AetRl lb polypeptide” meiides po I ypeptdes comics sing any naturally occurring polypeptide of an AetRJib family member as well ascny variants thereof (melnding mutants, fragments, fusions, and peptidomimetie formsphai retain a useRd activity. See, for example, WO/2006/012627. For example, ActRIfb polypeptides include polypeptides derived from the sequence of any known AetRIIb having a sequence at least about 80% identical to foe sequence of an AetRIIb polypeptide, and optionally at least 85%, 90%, 95%* 97%, 09% or greater identity. For sample,-an AetRlfoLpolypeplide oCthe invention may bind to and inhibit the function of an AclR I lb protein and/or activin. An AetRIIb polypeptide may be selected for:acti:y;iiyto promoting red blood cell formation in vivo. Examples of AetRIIb polypeptidesinclude human AetRIIb precursor polypeptide {SEQ ID MO; IS) arid soluble human AetRIIb polypeptides (e.g., SEQ ID NO: !6. 17, 20, and 21). The human AetRIIb precursor: protein sequence is as follows:

MTA PRV AEAIAttf G S EW PG $<3ltQERETiPiGl¥^tiA^1^I>EB^P|SS®X>SRO GESLlVLEAfWHiRHMPFYGMCDIHEDBGRPPPSmVOERPFQLEElK ARGPFGGVWKAQLNR DFVSQK ΪFPLQDKQSWQSEREIFS ΐ FGHKREE Lb Q FI AAEKRG S NLEVEXiWL ITAFH0KGS ET DYLEGNII TIMELC Η V AETM S RGLS YER:E D VP WC RSE GH K BS:I AH R DI'KSKR 7LLK S DATS VE ADEGm VPFE PGKPpG DTHGQ VG T RRYMAP EVLEGA IB FQR DA FEE I DR YABGX> V EWE L V S HGE MEG F VBEY ML P FEEBXGQB P SLEE LQE V V V B KKOH H PT IK DHWLKHPGLAOLeVTIEEChiDHDAEARLSAGCVEERVSLIRRSPBGTTS DCLVSLVTSWMVDLPPKESSI (SEQ ID NO: 15) lire signal peptide is single underlined; theextracellulacdornam is in bold and the potential bi-linRed glycosyiafion sites are in boxes·

The human AciRllb Soluble (extracellular), processed polypeptide sequence is as follows:

SGRGEAETREClYYMABWELERTRSSOLEPGEGEQDERLBGYASWSbSS GTI1LVKKGCWLDDFNCYDRQEGVAT EEMPQVYFCCCEGREGBERETBL PEAGGPEVTYEPPPTAPT (SEQ ID MO: 16) tie CAemnnal “talP of the extracellular dontam m Underlined The sequence with toe “tgjp deleted (a Δ15 sequence} is as follows:

^G'ReMTRECIYYmNW^.L^pTNQ^lhSRCEGEODKRLH.OYaSWAMg.S Gf IEtV'KKGCWLDDFKGTDRQ;SCW\T£EHfO^^^:SCSGNF€tilRFTHL P£A{SBQ ID NO: 17)

The nucleic acid sequence encoding a human ActRIIh precursor protein is as follows: (tsneieohdes 5-1543 of Genbank entry NM jOSI l§b)

ATGACGGCGCCeTGGGTGGeCCTCGGCCTeCTCTGGGGATeGCTGTGGe €CGGaTCTGGG€GTGGGGAGGCTGAGACAGGGGAGTGCATCTAGTAGM €G C€AACTGGGAGCTG GAG CGCACCAACCA6AGCG GGC TGG AG CGC TGG GA&amp;GG CGAGGAGGACAAGC GGG TGC AC TGGTAGGC CTC CT GGG C€ AAC A GCTCTGGCACCSTCGAGCTCGTGAAGAAGGGCTGCTGGCmGATGAeTT CAACTGCTAGGATAGGGAGGAGTGTGTGGCCACTfiAGGAG&amp;ACCCCCftG GTGTAeTTCTGGTGGTGTGAAGGCAACTTGTGCAAGGAGCGCTTCACTG atttgccagaggctgggggcgcggaagtcacgtacgagccaccgcggag AGCCCCCACCGTGCTGAGGGTGGTGGGCTACTCACTGCTGCeCATCGGG G GGC1PTTCCCTC ATG GT CCT GOT GGCCT TTTGGATG TACCG GCAT CG C A AGGCeCCCTAeGGTCATGTGGAeATCCATGAGGACCCTGGGCGTCCAGG AGCATGCCeTCTGGTGGGCCTGAAGCGftGTGCAGCTGCTGGAGATCAAG GCTGGGGGGGGGTTTGGCTGTGTGTGGAAGGCCCAGCTG?iTGAATGACT: TTGTAGCTGTGAAGATCTTCCCAGTCCAGGAGAAGCAGTGGTGGCAGAG' TGAACGGGAGATCTTCAGCAGACGIGGCATGAAGGAGGAGAAGCGGCTA CftGTTCATT:GGTGC€GAGARGGGAGGCTCCAA.CGTCG&amp;AGTAGAGCTGl GGCTCATCAGGGCCTTGCATGAeAAGGGSTCCCTGACGGATTACCTCAA SSGGAACATGATCAGATGGAACGAACTGTGTGATGTAGCAG AG ACGATG; tcacgaggcctctcatacctgcaigaggatgtgccctgstgccgtgggg aggggcacaagccgtctattgcccacacggactttAAaagtaagaatgt

AlTGCTGAAGAGCGAGCTGACftGCCGTGeTSGCTGACTTTGGCTTGGGT

GTTCGATTTGftGCCAGGGAAACCTCCAGGGGACACCGACGGBCAGGTAG

GCACGAGAGGGTACATSGCTCCTGAGGTGGfCGAGGGAGCeATCftftCTT

CCAGAGAGATGCCTTGCTGGGCAfCG^CATGTSTGCCATGGGGiTGGTG

CTGTGGGAGCTTGTGTCTCGCTGeAAGGCTGCAGACGGACCGGTGGAtfG &amp;GTACATGeTGe€efTTGaGGAAGAGATTGGCCAG€ACOCTTGBTTGGA GGftGCTGCAGGAGGTGGTGGTGCAeAAGAAGATGAGGCCGACeATTAAA GATCACT GGT TGA&amp;ACACC GG GGCCTGGC CCA GGTTTG TGT GAC CATCG &amp;GGAGTG C TG GGAGCATGATG CAGAGGGT CGC TTGCCCGCGGGC TGTGT GGAGGAGCGGGTGTCCCTGATTeGGAGGTGGGTCAACGGCACTACCTGG GAC’rGTCTGGTTTGGCTGGTGAGCTCTGTCAGCAATGTGGAGGTGCCeC CTAAAGAGTCAACH'iATCTAA (SEQ ID NO: 18}

The nucleic acid seq uenee encoding a human AcfEIIa selubl e #x trace! I elar) polypeptide is as follows: TGTGGGCGTGGGGAGGGTGAGACAGGGGAGTGGSTCTAeTACAACGCCA actgggagctgsagcgcaccaaccAgagcggcCtgGagcgctGcgaagg GGAGCAGGACAAGC GGG TGC AG TGGTACGC GTC CTG GGG CA&amp;CAGCTC ? gggaccatcgagctcgtgaagaagggctggtggctagatgagttcaact ggtaggatagggaggagtgtgtggccactgaggagaaggcccaggtgta CT TGTGCTGGTGT GAAGGCAAC ί TCTG C AACG AGBG CTTCACT CATTT G eCAGAGGGTGGGGGCCGGGAAGTGACGTACGAGCGAGGCCCGACAGCCC CCACC {SEQ ID NO: If}

Ip a speei&amp;: embodiment, the invention. relates to soluble ActRII polypeptides. As described herein, the term “soluble ActE II polypeptide” generally refers to polypeptides eouipr^mgip extracellular domain nf an AetRila or ActRIlb protein. T he term “solubfo Ael'RIlpplypeptide,” as used herein, includes any naturally occurring extracellular dotnam of an AetRila or AetRIib protein as well as any variants thereof lincluding mutants, fragments and pepfMermmeiieforms). An aetieindiinding AetRIl polypeptide is one that retains the abiisty to bind to activin, including, for example, aciivin AA, AB, SB, or forms that include a C or E subunit. Optionally, an activin-binding ActRII polypeptide will bind to activin A A with a dissociation constant of I nM or less. The extracellular domain of an ActKif protein binds to activin and Is generally soluble, and thus can be termed a soluble, aetivinfolnding ActRII polypeptide. Examples of soluble, aetl vin-binding AetRila polypeptides include tile soluble polypeptides illustrated In SEQ IB NOs: % 3,7,12 and I T, SEQ ID N0:7 is referred to as AeiRlla-hFe, and is described further in the Examples, Other examples of soluble, activin-binding AetRila polypeptides comprise a signal sequence in addition to the extracellular domain oCan ActRI l a protein* for example. the honey Reemellitm leader sequence (SEQ ID NO: 8), the tissueflaminogen activator (TEA) leader fSEQ II) HO: 9) or the native ActRIIa leader {SEQ ID HO: 10). The ActEIIa-hPe polypeptide illustrated in SEQ ID HO: 13: uses a TEA leader. Examples of soluble* actmnfomding AetRIIb polypeptides include live sotehle polypeptides illustrated in SEQ ID NOs: 16, 17, 20. Aetivinfomdlng AetRIIb polypeptides may also comprise a signal sequence in addition, to theiextoeelMar domain of an AetRIIb protein, for examples the honey bee mellttin leader sequence (SEQ ID NO: 8) or the tissue plarninogeh activator (XPAI leader (SEQ 1:0 ΝΘ: 9).

Functionally actrve Iragmehfsi of ActRIf polypeptides can be obtained by screening, pdlypepiides recombin tihtly produced from the eorrespondmg fragment of the nucleic acid encoding an ActRH polypeptide, In additidh, fragments can he chemically synthesized using techniques: kno wn in: the art such as convemional Merri field solid phase 0Mbc or t-Boe chemistry. The fragments can be produced (reepvnbiriamly or by chemical synthesis) and tested to identify those peplidyl fragments that can function as antagonists (inhibitors) of ActR 11 protein or signaling mediated fey achcim

Functionally active variants of AelRII polypeptides can be obtained by screening libran^ of modified polypeptides reeomhmantly produced tom tire corresponding mutagenized nucleic acids encoding an ActRII polypeptide. The variants cap he produced and tested to identify those that can Rmction as antagonists (inMhiiom) of AetRlTprotein or signaling mediated by activin. In certain embodiments, a functional variant of the ActRIIa polypeptides comprises an amino acid sequence that is at least 75% identical to an amlnp aeld sequence selected tom SEQ IDNOs: 2 or 3. In certain cases, the fencdional variant has an antino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical id an amino acid sequence selected from SEQ ID NQs: 2 or 3, I» certain embodiments, a functional variant of the ActRIIb polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected tom SEQ ID NDs: 16 or 17. In certain cases, the functional variant has an amino add sequence at least 80%, 8e%, 90%f:: :95%:i97%, 98%, 99%: or 1005¾ identical to an amino acid sequence selected from SEQ ID NQs: I T or 18,

Functional variants may be- generated by modifying the stmdureot an ActRI I ppIypbpR^dbr $»uh purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf HI© and resistance to proteolytic degradaiion in vivo). Such modified AciRil polypeptides when selected 10 reisih: aeti vio binding, are considered functional" equivalents - Of theTiaturaUy-occtimng ActR.fi polypeptides. Modified ActRIl polypeptides can also he produced, tor instance, by amino acid substitution, deletion, or addition. For instance, it is reasonable to; expect that:: an Isolated replacement of a leucine with an isoieucine or valine, an: aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino; acid with a structurally related amino acid (e.g.:, conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino aeids that arc related hi their side chains. Whether a change in the amino acid sequence of an ActRIl polypeptide results in a functional homolog can hereadily determined by assessing the ability of the variant ActRIl polypeptide:: to prodeee d response in eelld in afasMon similar to the wild-type ActRIl polypeptide^

In. certain embodiments;, the present invention contemplates specific raulationstof the ActRIl polypeptides so as to alter the glycosylation of the polypeptide. Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-lirtked OrM-lihkediglyeosylation cites. Asparaginedkdred glycosylation recognition sites generally comprise a tripephde sequence, asgaragine-Xdhredmne or asparagine~X~serine {whet© id any aniino acid) which is specifically recognised by appropriate cellular glyeosylation enzymes. The alteration may also fee made fey the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ActRIl polypeptide (for lirtked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or mird amino acid positions of a glycosylation recognition sitefsnd/or amino acid deletion at the second position) results in non-giyeosylaiiou at the modified iripeptide sequence. Another means of increasing the number of carbohydrate moieties on m ActRIl polypeptide is by chemical or enzymatic coupling of glycosides to the ActRIl polypeptide. Depending on the coupling mode used, the sugaffs) may be attaebed to (af arginine and histidine) (b) freecarboxyl groups; (c) tree sulthydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of seri ne, threonine, or hydrpxyproli ne; fe) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine, Removal of one or more carbohydrate moieties present on an AetRIl polypeptide may be accomplished eheniically and/or enzymatically. Chemical deglycosyiatson may involve, for example, exposure of the ActRIl polypeptide to tbe compound irifiuoromeihlfiesulfojye qeid,/drah equivalent compound. This treatment results in the cleavage of most or all sugars: except: the linking sugar: (1M^acety!gi»eosa.mlne: or N-icetflgalaciosamine), while leaving the amino add sequence intact Enzymatic cleavage of carbohydrate mqlefids .on .ActRll polypeptides can be ach ieved by the use of a vari ety of undo- and exo-'glyeosldases as described by Thotakuraet ail, (1987:) Meth, BnzymOh.138:350: The sequence of an ActRll potypeptide: may: he adjusfedj.as·· appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected fey the amino acid sequence of the peptide. In general, ActRll proteins for use in humans may fee expressed in a mammalian cell line that provides proper glyeosylation, suefe as BEK293 or GHp cell lihes, although other mammalian expression cell lines are expected to he useful as well.

This disclosure further contemplates a method of generating mutants, particularly sets of combinatoric mutants of to ActRll polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries may be to generate, lor example. ActRI I polypeptide variants which bind to activin or other ligands. A variety of screening assays are provided below, and such assays may fee used to evaluate variants. For example, an ActRll polypeptide variant may: be screened for ability to bind to an ActRll ligand, to prevent bindingof an AeiRli ligand to an ActRll polypeptide or to interfere with signaling·caused.fey an AetRlI ligand.

The activity of an ActRll polypeptide or its variants may also be tested in a eelRbased or in vivo assay. For example, the efleet of an ActRll polypeptide variant on the expression of genes involved in hematopoiesis may be assessed. This may. mi needed, be perfbnned in the presence of one or more recombinant ActRll ligand proteins (e.g,,: activin). .and cells may fee transfected so as to produce an. ActRll polypeptide and/or variants: ihereofLtod: Optionally, to: AetRllligand. Likewise, an ActRll polypeptide may fee administered to a mouse or other anith%artdl;ohe,rir-triore .Blood meMurefnent$, such as an RRC count, hemoglobin, or reticulocyte cooptotay be assessed^

Combmaiorially-derived variants can be genetefed which have a selectiveor generally increased potency relative to a naturally occurring::ActRll polypeptide. Likewise,: mofagenesisean give rise:to variants which have intracellular half-lives dramatically different than the;corresponding a wild-type; ActRII: polypeptide- For example, the altered protein can he rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or ofoerwise inactivation of a native AetRII polypeptide. Such variants, and the genes which encode them, can he utilized to alter AetRII polypeptide levels by modulating the halfolife of the AciRil polypeptides, for instance, a short halMifo can give rise to more transient biofegic#;^ecis:5®drwhm:.^tt of an inducible expression system^ can allow tighter control of recombinant AetRII polypeptide levels within the celly in an Pc fosion gmtein, mutations may be made in the linker (if any) m&amp;at the Fe portion to alter the hall-life of the protein.. A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which: each include at least « portion of potential ActR.1l polypeptide sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such; that the degenerate set of potential AciRO polypeptide: nucleotide sequences are expressible as individual polypeptides, or alternatively^ as a set of larger fusion proteins (wg., for phage display).

There are many ways by which tlmiibrary of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in m antotnatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is wdl known in the art (see lor example, Narang, SA (1983} Tetrahedron 39:¾ Itakura et ah, (19S1) Recomhlnant ΙΤΝΑ, Free. 3rd Cleveland Sytnpos,

Maoromolectdes, ed. AO Walton. Amstcrdarn: Elsevier pp273~2H9; Italcnra et ah, (19§4) Annu. Rev, Biochem, S3:323;ltakura et ah, (1984) Science l9&amp;:1 056* lke;iet-zt.>/(i 983) Mucleie Add Res. ! 1:477). Such techniqucrhave been employed in the directed evolution of other proteins (see, for example, Scott et ah, (1990) Science 249:386-390; Roberts et ah, (1992)MAS USA §9:2429-2433; Devlin et ah, (1990) Science 249; 404-406; Ewiri a et ah, (1990) FMAS USA ST; 6378-6382; as well as US. Patent ISfos; 3,223,409,5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, ActfUi polypeptide variants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Rufet ah, (19941 Biochemistry 33:1565-15?2; Wang et aL (i994} j. Biot. Chem. 269:3095-3099; Balini et at, (1993) Gene 137:109-1 IS; Grodfeerget at, (1993) Bun 3, Bioelmm, 218:597-601; Nagashrma et at, (1993) 3. Biot Chem. 268:2888-2892; Lowman et at, (1991) Biochemistry 30:10832-10838; and Cunningham et at, (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et at, (1993) Virology 193:653-660; Brown el at, (1992) Mol, Get! Biot 12:2644-2652: McRriight et at, (1982) Science 232:316); by saturation mfoagenesis (Meyers ei at, (1986) Science 232:613); by PGR mutagenesis (Leung et at, (1989) Method Cell Mo! Biol 1:1 !-!9);of by random mntogenesis, including chemical mutagenesis, etc. (Miller et at, (1992) A Short Gonrse in Bacterial Genetics, CSHL Press, Gold; Sprmg Harbor, HV; and Greener et at, (1994) Strategics in Mol Biol 7:32-34). Linker scanning nwiagenesis, particularly in a oombmatoriai'4i^iit%:iS'''aft Miractive method for identifying Ironeated (bioactive) forms of ActRIi polypeptides, A wide range of techniques are known in the art for screening gene products of oonfomatorial libraries made by point mutations and truncations, and. for that matter, for screening eBMA libraries for gene products having a certain property. Such techniques will he generally adaptable for rapid screeningof the gene libraries generated by the combinatorial mutagenesis of AetRl! polypeptides, The mosi 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 of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Preferred assays include aetivin binding assays and aetivin-mediuted ceil Signaling assays.

In certain emhodimeats, the ActRii polypeptides of the invention may forther comprise post-translational modifications in addition to any that are naturally present in the ActRii polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, giycosylatlon, phosphorylation, lipidatiOo, and acylation. As a result, the 'modified ActRI! polypeptides may; contain non-amino acid dements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the fonetfonality ofah ActRii polypeptide may be tested as described herein for other ActRI! polypeptide variants. When an ActRI 1 polypeptide is produced in cell® by elpyirig a nascent form of the Act&amp;Ii p'dlypejpti^ post-translational processing may also fee important. for conoct folding and/or fonetion of foe protein. Different cells (such as CMO, BeLa, MDCK, 293,,Wf3% N1H*3T3 or MBK293) have specific cellular machinery and characteristic mechanisms for such post-tmnslaiiooal activities and may he chosen to ensure the correct modifieatfotl and processing ofthe AeiRll polypeptides.

In certain aspects, functional variants or modified forms of foe AMRO polypeptides include fusion proteins having at least a portion of the ActRIt polypeptides and one or more fusion domains. Well known examples of such .fusion domains include, but are not limited to, polyhistidine, Olu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein 0, ah imthnnoglobiilin heavy chain eohstant region (Fe), Maltose binding protein (MBP), of human serum albumin, A fusion domain may he selected so as to confer a desired property. For example, Some fusion domains are particularly useful for isolation of the fusion proteins by afhnity eltromaipgraphy. For the purpose Of affinity purification, relevant matrices for aflnity ehromaiogMphy, such as glutathione-, amylase-, and nickel- or cohalt- conjugated resins are used. Many of such matrices am available in "kit” fort», such as the Pharmacia GST purification system and the QIAmtpnsss™ system (Qiagen) nseful with (FI1S&amp;) fusion partners* As another example, a fusion domain may be selected so as to facilitate detection of the ActRIl pplypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., <3FP) as well as '‘epitope tags/' which are usually short peptide, sequences for which a specific antibody is available^ Well known epitope tagsMr which specific monoclonal antibodiesare readily available include FLAG, infiuenzs virus haemagglutinin (H A), and eonye tags, '.In some eases, the fusion domains have a protease cleavage site, such as for Factor Xa or Ihmmbin, which allows the relevant protease to partially digest the fusion proteins and thereby libefofo tlm mcomhinani proteins therefforn. T he liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, an AetRlf polypeptide is fused with a domain that stabilizes the ActRIl polypeptide in vivo (a “stabiltzef· domain). By "'stabilizing55 is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or Other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known fo confer desirable pharmaeokinetic properties on a wide range of proteins, Likewise, fosions to hurnan serum albumin can confer desirable properties, Other types of fusion domains that may be selected include multimeriking (e,g.. dimermng, tetramerimng} domains and.functional domains (that eonfer an additional biobgioat funeiion^ such as further stimulation of muscle growth).

Mb a specific example, dm present invention providesa felon protein comprising a soluble extracellular domain of AetROa feed to an Fe domain SEQ ID hlO: 6). THT C FPC F A P E LLGG P$ V EL E P FK P K OTEHIS FT P EVTC¥VW {A) ¥SBED FEYK FMW Y Y DS;

¥E¥HmKTKPREEQYKSTYRWSVLT¥feSDWLdGKE¥KC^ iEifSflKftEFVfeEKTISKSK GaPREPQVYTLPPSREEMTRKQVSLT€L\?RGF¥PS0IWS5?ESRG^PEHNYKTTFPYLDSDG ΡΕΕΕΥ8Κ1.Τν0Ε8ΚΜ^β^¥ΕΒ0$ν^ΗΕΑΡΗ§:(^} HYTQKSLSLSPGK*

As an additional spemfie exampiey fe present Invention provides a fusion protein comprising a soluble extraeellnlar domain of ActR I lb fused to an Fe domain (e g., SEQ ID .NO: 21).

SEEGEAETHEClYYtlAriREEERtNQSGLESCEGESDKRLHDYASWAtlSSGTrEEVKRQCKLD

DFNCYDEOSGYATEEN PQVPECCCEGGEGΜΕΕΡΤΗΙ» PEAGGPEVTYEPPPTAPTGGGTMf€ P PC PAPELLGG PS'VFLPPPKEKDYLMISET PE¥YC¥F¥DYSHEDPEVEFGWYVDGVEYHiAKT

ΚΡΚΕΕ0ΥΝ3ΤΥΗννΕ¥ΕΤνΕΗ:<2Ο^ΕΝσΚΕΥΚΕΚ¥8ΕΕΑΕ:ΡνΡΕΕΚΤΪ$ΚΑΚ6αΒΡΕΡανΥΤΕ PPSREEMTKEQVS LTGLY KG F Y P S D1A V EWSSMGQ P EGE YRTT PPVLD S DG S F F E;¥ S KLTV D K:3RWQQGN: V EE GSYM Η E A LEE H YTQKSESL S PG R

Optionally the Fe domain has one or more mutations at residues such as Asp-265,, feme 222*and Asn-434, In certain cases* the mutant Fe domain having one or tuore of these mutations («.g,, Asp-265 mutation) has reduced ability of binding to the Fey receptor relative to a wildtype Fc domain, in other cases, the mutant Fc domain having one or more of these mutations (e.g., Asn-434 muiationj:has increased abili ty: of binding to the MHO class I-"rdated Pc-receptor (FcRN) relati ve to a wild type Pc domain.

It is understood that dirierent elements: of the felon proteins may be arranged in any manner that is consistent with the desired functionality. For example, an ActRII polypeptide may be placed C-temrinal to a heterologous: domain* or*, alternatively, a heterologous domain may be placed C-termioal' to: an ActRII polypeptide. The ActRII polypeptide domain and the heterologous domain: need not be adjacent in p: feiod prptein, and additional dofeiosor amino acid sequences may be included 0- or HAermlnalto either domain or between the domams:

In eeftain embodiments, the ActRIl polypeptides Of the presehi invention, corstam one: of more modifications that are capable of stabillzih|:ihe: A:etllil polypeptides. Eor example, such' modifications enhance the in vim half life of the ActRIl polypeptides, enhance circulatory half life: of the ActRIl polypeptides or reducing proteolytic degradation of the AciRII pplypeptidos. Snob stabilizing modifications Incinde, but are not limited to;,, fusion proteins (includingj for example, fusion proteins comprising an AefRII,polypeptide and a stabilizer domain), modifications of a glycosylafien site (including, for example,: addition of a glyeosyl alien site to: an; ActRIl polypeptide), and modifications; of oarbohydrgte moiety (including, for example, removal of carbohydrate moieties from an ActRIl polypeptide). An used herein, the fend “stabilizer domain” not:only refers to a fusion domainp.g,, Fc) as in the case of fosion proteins, but also includes nonprpteioaoepas modifications speh asp: Oarbohydrate nioiety, or nonproteinaceous moiety, such as polyethylene glycol.

In certain embodiments, die present invention makes available isolated and/or purified forms of the ActRI l polypeptides, which are isolated from, or otherwise substantially free of, other proteins, ActRIl polypeptides will generally be-prodneed by expression from recombinant nucleic acids, 3. Nucleic Acids Encoding ActRIl Polypeptides

In certain aspects, the invention provides isolated and/or recombinant nucleic acids encoding any bffoc AciRII polypqptidos (o,g,, soluble ActRIla polypeptides and soluble Actiilb functional variants and fusion proteins disclosed herein. For example, 5Ε01Ϊ> ΜΟ> 4 encodes the naturally occurring human ActRIla precursor polypeptido, while SRQ ID NO; 5 encodes the processed extracellular domain of ActRIla, For example, SEQ ID NO; IS eneodes the iaaturaity occun inghuntan AetRllb precursor polypeptide, while SEQ ID NO: 19 encodes the processed extracdlular domain of : Act RI lb. The suhieet n ucieie acids may be s i ngfe-stra nded or double: strand ed. Such nncl eie acids may be DN A or RMA molecules. These nuciem acids may be used, for exaptple, in methods for rnaking AofRll polypeptides or as direct therapeutic agents (eg., in a gene therapy approach).

In certain aspects, the subfect nucleic acids encoding AetRliapolypeptides are forther understood to include nucleic adds that are variants of SEQ ID NO: 4 or 5, in certain aspects* the subject nucleic acids·:.encoding Actiph polypeptides ore further understood to ioclpde oueleic adds that are variaiiiis of S1Q ID NO: 18 or 19. Variant nucleotide sequences include sequences that diiler by one or more nucleotide substitutions, additions or deletions, such as allelic variants.

In certain. embodiments, theinvention provides isolated or reeombmaiii nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 4, %. IS, or 19. One of ordinary skill m the art will appreciate that nucleic acid sequences complementary to SEQ IDNOsld, 5, '18:, or 1¾ and variants of SEQ ID NQs:4,5, 18, or 19 are also within the scope of this invention. In Ihrthcr embodiments, the nucleic acid Sequences of the invention can he Isolated, recombinant and/or fused with a heterologous nucleotide sequence, or in a ON A library.

In other embodiments, nucleic acids of the invention also include: nucleotide sequences that hybridize under highly stringent conditions to the mmieotide sequence designated in SEQ ID NOs: 4, 5,11, or 19, complement sequence of SEQ ID NOs: 4, 5, 18, or 19, or fragments thereof. As discussed above, one of ordinary skill in the art will understand readily that appropriate riringency conditions whichpromote DMA hybridization can be: varied. One Of Ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at: &amp;0 x sodium chi oride/sodiunr citrate (SSC) at about 45 °C, followed fey a wash of :2,0ix: SSC at 50 *€: For example, the salt concentration in the wash step can be selected .fount O' low stringency of about 2,0 x SSC at 50 -C to a high stringency of about 0.2 x SSC at 50 %% M addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 to high stringency conditions at about OS Both temperature and salt may fee varied, or temperature or salt concentration may fee held constant while the ether variable is changed. In one embodiment, the invention provides: nucleic acids which hybridize under low stringency conditions of 6 x SSC at room: temperature followed by a wash at 2 x SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forth' in SEQ ID NOs: 4, 5, 18, or 19 due to degeneracy in the genetic cod© are also within the scope of the invention. For example, a number of amino acids are dcslghated fey more than one .triplet. Codons that specify the same amino acid, or synonyms (for example, DAU arid CAC are synonyms for histidine) may result in:“silent” mutations which do not affect the amino acid sequence of the protein. However, it is expected that DMA sequence polymorphisms that do lead to changes ih the amino acid seqnMces of the subfect proteins will exist among mammalian cells. One skilled in the art: will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nneteotMesf of the oueleic aesds encoding a particular protein may exist among individuals of a given species due to natural allelic variation, Any and all such nucleotide variations and resultingamino acid polymomhisrbs are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids of the invention may be operafely linked: to one or morefegolatory nuelpotide sequences in an expression construct. Regulatory nucleotide sequences will generally: be appropriate to the host pel! used for expression, Numerous types of appropriate expression vectors and suitable regulatory sequences are knowii m the art 'lot a variety of host cel ls. Typically, said one or mpre regulatory: nucleotide sequences may include, but are not limited to, promoter Sequences, leader or signal sequences, ribosomat binding sites, transcriptional: start and ferndnation sequences^ translational start and terminatipn sequences, and enhancer or activator sequences, Constitutive or inducible promoters as ktMWn in the art are contemplated by the invention. The· promoters: may be either naturally occurring promoters, or hybrid pmmoters that combine elements of more than one promoter. An expression eonstrnet maybe present in a 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 used.

In certain aspects of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding ah AeiRf l polypeptide and uperably linked to at least one regulatory sequence. Ilegulatory sequences arc art-recognized and are selected to direct expression of the ActRO polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gme-'^fressim Techmbgg*. Methods in Enzymology, Academic Press, Sap Diego, €A (19%). For instance, any of a wide variety nf expression control sequences that control the expression of a DNA sequence when operatively linked to it may he used in these vectors to express DSA sequences encoding an ActRll pol peptide. Such useful expression control sequences, include, for exmaple, the early and late promoters of tet promoter*. adenovirus or cytomegalovirus immediate early promoter, RSV pt^poters, the lac sysfohg the trp system, the TAC or TEC system* T? promoter whose expression is directed by T7 RMA polymerase* the major operator and promoter regions of phage lambda, the control regions for id coat protem, tbe promoter for 3-phospbogiyeerate fcjnase or other glycolytic enzymes, the promoters of acid phosphatase* &amp;g., PhoS, the promoters of the yeast a~mating factors, the polyhedron promoter of the baculovlrus system and other sequences known to eonpoi the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof It should be understood that the design of ihe expression yeetor may depend on such factors as the choice of the host ceil to he transformed andfordhe type of protein desired to he 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 be considered, A recombinant nucleic acid of the invention can he produced by ligating the cloned gene, or a portion thereof into a vector suitable for expression in either prokaryotic cells, eukaryotic ceils (yeast, avian, insect or mammahanj, or both. Expression vehicles for production of a recombinant AetRO polypeptide include plasmids and other vectors. Tor instance* suitable vectors include plasmids of the types: pBR322~derived plasmids, pEMEL-derived plasmids, pEJCfoerived plasmids, pRTac-derived plasmids and pllC~derived plasmids for expression in prokaryotic cells, such as E, coii, home mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that arc expressed in eukaryotic: cells. The pefEvAl/amp, pcDNAI/neo, pEc/CMV, pSV2gpt, pSV2neo, pSV2-dhff, pTkS, pRSVneo, pMSO. pSVT?, pko-neu and pHyg derived vectors are examples of mammalian expression vectors sui table for transfection of eukaryotic cells. Some of these vectors are modified with sequences fmm bacterial plasmids, such as pBR322, ΐ0>facilitate replication anddfug resistance selection in both prokaryptic and eokaryotiecells. Alleriiatively, derivatives of viruses such as the bovfnepapiiiema virus (BPV-!}, or Epstein-: Barr virus CpfiBBo, pREf-derived and p20|) can be used for transient expression of proteins: hveukaryotie ceils. Examples of other viral (including retroviral) expression systems can he found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids aud io transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular CMnlngA laboratory Manual, 3rded. by Sambrook, Fritseh and Maniatis (Cold: Spring Harbor 'laboratory Press, 2001). In some instances, it may feeidesirable to express the recombinant polypeptides by the use of a baeuloviras expression: system. Examples of such baeuloviras· expression systems include pYLfoerived vectors (sueh as pVLl392:i :pVL13P3 and pVLPdl), pAeU W-derived vectors (such as pAeUWl), and pElueBae-derived vectors::(such as the fogal containing pBlueBac 111).

In a preferred embodiment, a vector will be designed for production of the subject ActS.II polypeptides in CHO celts* such aa a Pemv-Serigt vector (Stratagene, La Jolla, Calif), pcDfiAd vectors (Invitrogen, Garlsbad, Calif) and pGTneo vectors (Promega, Madison. Wise.), As will be apparent, the subject gene constructs can be used fo cause expression of the subject AetRII polypeptides in cells propagated In culture, e,g., to produce proteins, including fosion proteins or variant proteins, for puriication.

This disclosure also pertains to a host cel! transfected with a recombinant gene including^ coding sequencefe.g.jSFQ ID MO: 4, 5, 18, or 19) for one or more of the subject AetRII polypeptides. The host cell may be any prokaryotic:or eukaryotic cell* For example, an ActRH polypeptide of the in vention may be expressed ip bacterial cells such as E, coll, insect cells (e.g,, using a baeulovims expression system), yeast, or mammaliap eel|s> Other suitable: host ceils are known to those skilled in the art:

Accordingly, the present invention fortber pertains to methods of producing the subject AeiRII polypeptides, for example, a host ceil transfected with an expression vector encoding an ActRlla or ActRllb polypeptide car? be cultured under appropriate conditions to allow expression of the ActRH polypeptide to occur. The ActRH polypeptide may be secreted and Isolated from a mixture of cells and medium containing the" ActRH polypeptide. .Alternatively,, the AetRII polypeptide may be retained eytopiasmlcally or in a membrane fraction and the eel Is harvested, lysed and the protein isolated. A ceil culture includes host cells, media and Other byproducts. Suitable media for cell culture are well known in the art.

Tiis subject AetRll polypeptide? cap be isolated front: cell culture medium,: host cells, or both, using techniques known nr the art for purifying proteins, including ion-exchange chromatography, get filtration chromatography, uitrafiiiration, electrophoresis, iranmioaffifiify purification with antibodies specific:: for pariicuiar epitopes of the ActRIi polypepudes and affinity purification with an agent that binds to a domain fused to the AetRll polypeptide (e.g., a protein A column may be used to purify an ActRIla-Fc or AetRllb-Fc fusion), in a preferred embodiment, the Ac® Π polypeptide is a fusion protein containing a domain which facilitates its purification. In a preferred embodiment,: purification is achieved by a series of column ebromatopaphy steps, including, for example, three or more of the following, in any order: protein A ehrosmdography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography, The purification could be completed with viral filtration and buffer exchange. As demonstrated herein, ActRlla-hFc protein was purified Ip a purity of >98% as determined by size exclusion chromatography and >95% as determined by SPS PAGE: This level of purity was sufficient ip achieve desirable results in mice, rats and non-human primates.

In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinasc cleavage site sequence at the N~terminus of the desired portion of the recombinant : AetRll polypeptide, can allow purification of the expressed fusion protein by affini ty chrom atography using; a Ni2:i’ metal resin,:.' The 'purification leader sequence can then be subsequently removed by treatmeht with enterokinase to provide the purified AetRll polypeptide{e,g,, see Mochuli et al, (Ι987|R Chromatography 411:177; and fanhhecht et al, Pit AS USA 88:8972).

Techniques for making fusiort genes are well known. Essentially, the joining of various 0!riA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing bluntrondeder stagger-ended termini for ligation, restriction ehz>a«e digestion to provide: for appropriate: termini, fi!!sng~in of cohesive ends as: appropriate, alkaline phosphatase treatment: in avoid undesirable joining, and enzymatic 1 igation. In another embodiment, the:fuston gene can be synthesized by conyeniipnal techniques including antomatediDNA syttihesizetis. Alternatively. PCR amplification of gene fragments can be carried out using anchor primers which give rise to compfenientary overhangs: between two cofiseeptiye gene fegiyJents which can subse^^htly be annealed to generate a ehimeric gene sequence (see, for example.. Current Protocols in MoieeipAr Mokigy. eAs, Ausohcl et ah. John Wiley &amp; Sons: 1992}*

4. Mtofflatiye,MtMM TKedata presented het^tn demonstraies that antagonists of sctivio-AetRll signaling can fee used to increase red blood cell or hemoglobin levels, Although soluble ActRlIa and AetROb polypeptides, and particularly ActRIIa-fc and AetRlib-Fc, are preferred antagonists, and1: although such antagonists may affect red bipod cell levels :¾ meebpisha- ©thef than activin antagonism (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 the TGF-beta superfemily, and soch ebllective inhibition may lead to the desired effect on hematopoiesis}, other types of aetivin~AetRn antagonists arc expected to he useful, including anti-activin (e.g., activin pA, ifo pc and βρ) antibodies. anti-ActRUa antibodies, anti~AetRllb antibodies, antisense, RNAifor fifeozyip©nuclei© #jds ih$ inhibit thepifoductiGn of ActROa and/of ActRilfe, and other inhibi tors of activin, AetROb or AotRIISj partieniarly those that disrupt activin-AetRila and/or amivin-ActSIlh binding.

An antibody that is speeihcally reamive with an ActRIf polypeptide (e.g-, a soluble ActRlIa or AeiRilfe polypeptide) and which either binds competitively to ligand with the ActRII polypeptide or otherwise inhibits AciRO-mediated signaling may be used as an antagonist Of AetRIl pdlypeptide aetivities. Likewise, an antibody that is speeiRcally reactive with an activin βa, Pa, Pc or (¾ polypeptide, or any heterodhner feereof, and which dismpts ActRlIa atid/or AetRIlb binding may he: Used as an antagonist.

By using immunogens derived from an ActRlIa polypeptide, AetROb polypeptide or an activin polypeptide, anti-proteio/anfopeptide antisera Of monoclonal antibodies can he made by Standard protocols (sec, for example, Antibodies; A Laboratory Manual ed, by Harlow and Lane (Gold Spring Harbor Press: 1988)}. A mammal, such as a mousey a hamster or rabbit can be immunized with an immunogenic form of the activin, ActRlIa or AetRIlb polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or n fusion protein,. Techniques: for conferring: rmmunogenicity on a protein or peptide include con)ugatibn to carriers or other techniques well known in the art. An immimogenlc portion:: of an ActJlil or activin polypeptide cars be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or semni. Standard ELISA or other hnmonoassays can be used with the immunogen as antigen to assess the levels of antibodies.

Following horounizafionof an animal with an antigenic preparation of an activin, AetRlia or ActRIlb: polypeptidev antisera can he obtained and5 if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing eelis: (lymphocytes) can be harvested from an immunized annual and fused by Standard somatic ech fusion procedures with immortalizing cells such as tnyeiotna oells to yield hybridoma cells. Such teehhtcgues are well known in the prt, and include, for example, the hybridoma technique (originally developed by ICohler and Milstein, (19?:l)^atpiP, 256t 495-497), the human R cell hybridoma technique (Kozhar et al,, (1983) Immunology Today, 4: 72:), and the ER^-hybridomadeetmitiue to produce human monoclonal antihoilips (Cole et aL, /(1985} Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-90). Hybridoma eelis ean be screened immunochemieally for production of antibodies: specifically reactive with an activin, AotRila or ActRIlb polypeptideahdbwnoelonal antibodies Isolated ffoin a culture comprising such hybridoma cells.

Tire:i^*R7<antjbody9?M used hereinis intended to include whole antibodies, e.g,, of any isotype (IgG, IgA, IgM , IgE, etc), and includes fragments or domains of immunogiobullns which are reactive with a selected antigen. Antibodies can be fragmented using conventional teehmqnes and the imgjnents screened for inility and/or interaction with a speeific epitope of interest. Thus, the term includes segments of proteolytteally-eleaved or recomhinantly-prepared portions of an antibody mofoonlethai are capable of selectively reacting with a certain protein. Mon-l imiting examples of such proteolytic and/or recombinant ifa^nents inelude Eab, F(ah’)2, Fab', Fv, and single chain antibodies fscFv) conlaining a V[L| and/or M[M] domain joined by a peptide linker. The scFv's may he covalentiy or noh-eovalentiy linked to form antibodies having two or more binding sites. The form antibody also includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies. The term Aecdrobinant antibody^, means an antibody, or antigen binding domain of an sntmunoglabphu, expressed from a nucleic acid that has been constructed using the techniques, of molecular biology, such as a humanized antibody or a fully huma«;Wtifeoiiy developed from a single chair; antibody. Single domain and single chain antibodies are also mchfoedwithm the term “recombinant antibody”. in certain embodiments, anantibody of the invention is a monoclonal antibody, and in certain rnibodim^t$v%!ginvmiioii::mak€S''avatla6le''iB^tod?,fe;f^«»«s!d»g novel antibodies. For example, a metlpd for generating a rhonoclonal antibody fffet hinds specifkallyto an AeiEIla polypeptide, AefRIlb polypeptide, or aotivin polypeptide may comprise administering to a mouse an amount of an immunogenie composition comprising the antigen polypeptide effective to stimulate a detectable immnne response^ obtaining antibody-producing cells (eap, cells from the spleen) .the antibody- producing cells with rnyefoma cells to obtain antibody-producmg hyhridomas, and testing the antibody-produemg hybridomas to idepity a hyhndprna that produces a monocolonai antibody that hinds specifically to the antigen. Once obtained, a hybridoma can he propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifreally to the antigen. The monoclonal antibody may he purified from the cell culture;

The adjective “specifically reactive with” as used in reference to an antibody is intended to mean, as is genemlly understood in the aft, that the antihody is sufficiently selecti ve between the antigen of interest {e,g„ an aetivin, ActRIla or ActRIlb polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample, in certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross-reacting polypeptides. One characteristic that influences the specificity of an anfbody:antipn interaction is the affinity of the antibody for the antigen. Although the desired specificity may be reached with 8 rangepFdifferent affnities, generally preferred antibodies will have art affinity (a dissociation constant) of about 1CF6, 10", 10 s, 10' M or less.

In addition, the techniques used to sefoen antibodies in order to identity a desirable antihody may influence the properties of the antihody obtairsed. For example, if an antibody is to he used for binding an antigen in solution, it may be desirable to test solution hind mg. A variety of different techniques areavailaMe for i int^r^otiQ^ l?etwfeen antihodiesund antigesis t© identify particularly desirable-antibodies, Sack techniques include £LI$A4 surface plasmon resonance binding assaysf&amp;g., the Biaeore^ binding assay, Biaeore AB, Uppsala, Sweden), sandwich assays (e,g.s the paramagnetic bead system of 1GEM International, Inc., Gaithersburg, Maryland), western blots, hmnunopmcipitation assays, and immunohistochemistry. f&amp;amples of categories of nucleic acid compounds that arc aetivin or AetRO antagonists include antisense nucleic acids, RMAi constructs and catalytic nucleic acid consiruetsi A nucleic acid compound may be single or double stranded, A double stranded compound piay also include regions of overftang or non-complementarity, where ode or the other of the strands is single stranded , A single stranded compound may include regions of sefiAomplementariiy, meaning that the compound forms aso-eaUed structure, with a region of double helical structure. A nuo!^o<a0td:.^m|found:iday:^mprise.d: nucleotide sequence that Is complementary to a region consisting of no more than 1000, no more than :300, no more than 250, Up more than ! 00, or no more than 50, 35,23,22,20,18 or 15 nucleotides of the full-length ActRii nucleic acid sequence or aetivin fh^, 0b, pc* or Pe nucleic acid sequence. The region of coinpiementarity will preferably be at least 0 nuclebtides, and optionally about 18 fo 35 nucleotides, A region of complementarity may fall Within an intron, a coding sequence or a noucodlng sequence of the target transefipi, such as the codi ng sequence portion, Generally, a nucleic acid compound will have a length of about E to about 300 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides, A nucleic acid may be a DMA fparticularfyior use as an antisense), RMA or RN A.DNA hybrid. Any one strand may include a mixture ofiDNA and ENA, as well as -modified forms that cannot readily be classified as either Obi A Of RMA. Likewise, a double stranded compound may be D14A:DNA, DfNAtRNA or RHA:RNA, and any one strand m ay also incl ude a mixture of ON A and RN A, as well as modified forms that cannot readily be classified as Gthcr DNA or ENA. A nucleic aeid compound may include any of a Variety of modi fieatioos;, -including one or mod ifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including intemucieotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). An antisense nucleic acfd'eompound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability m the: serum, in a cell of:in a 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 construct, the strand complementary to the target transcript will generally be ENA or modifications thereof The other strand may be ENA, DMA or any ether variatiom The duplex poriion of dottble stranded or single stranded ‘ΤύΑίΓρΙη^ RlNAi construct will generally have a length of 18 to 40 nudeotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate. Catalytic or enxyrnatie nucleic acids may be ribozymes or DNA enzymes and may also contain modified forma. Nucleic acid compounds ;may in3rib.it expression of the target by about ;5Q:%, 7S%3 90% pfmore when contacted wi th ceils: under physiological conditions, and at a concentration where a nonsense or sense control hp little dr no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and IE micromolar, Nucleic acid compounds may also be tested for effects On, for example red blood cell levels. 5. Screening Assays in certain aspects, the present invention relates to the use of ActEE polypeptides (c.g>, soluble AetEl la or AetRIlh polypeptides) and aetivin polypeptides to identify compounds fogeuts) which are agonist or antagonists of the activin-AcfEIIa and/or aetivin AetRIlh signaling pathway. Compounds identified through this sereening can he tested to assess; their ability; to modulate red blood cell, hemoglobin and/or reticulocyte levels in vivo or in vitro. These compounds cap be fested, for example, in animal models.

There are numerous approaches to screening for therapeutic agents for increasing red blood cell or hemoglobin levels by targeting actfym and ActRfl signaling. In certain embodiments,; hi^-throughput screening of compounds can be carried put to identify agents that perturb aetivin or AetRITmediated effects on a selected ceil line. In certain embodiments, the assay is carried out to screen and identify compounds that speeiheally inhibit, or reduce binding Of an AeiRIIa or AetRIlh polypeptide to aeti vim Alternatively, the assay can be used to identify compounds that enhance binding of an AetEila or ActE I lb polypeptide to aetivin. In a further embodiment, the compounds cun be identified by their ability to interact with an aetivin, ActE I lb polypeptide, or AciRlla polypeptide. A variety of assay formats will suffice, and,, in tight of foe present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in foe art. As described herein, foe test compounds (agents) of foe inventionmay be ereated hy any combinatorial cheroic al method. Alternatively, the subject compounds may be naturally occurring biontolecules 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 forample, by baeteria, yeast, plants or other organisms (e.g., natural products), produced chemically (e g., small molecules, including peptidQMifoeties|, or produced reeombinantly. Test compounds contemplated by foe present Invention include non-peptidvl organic molecules, peptides, polypeptides, pepiMomimeties, sugars, honnones, and nucleic acsd molecules. In a specific embodiment, the test agent is a small organic molecule having a molecular weight of less than abont 2^000 Daltons.

The test compounds of foe invention can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry, These libraries can compose, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Preseatahoo of test compounds to foe test system can be in either an isolated form or as mixtures of eompounds, especially in initial screening steps. Optionally, the compounds may be optionally denvatized wifo other compounds andhayedeti^ahfong groups that facilitate isolation of the compounds, Mon-limiting examples of denyatizing groups include biotin, fluorescein, digoxygenin, green fluorescent proteim ispfopes, polyhistidine, magnetic heads, glutathione B; transferase (GST), pkofoactivaiihfe crosslinkers 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 surveyed in a gi ven period of time. Assays which are performed in celbfl'ee systems, such as may be derived with purified or semi-purified proteins, are often preferred as '’'primary" screens .fo that they can be generated to: permit rapid development and felatively easy detection of an alteration in a molecular target which is mediated by a f est compound. Moreoveri foe effects of cellular toxicity or bioavsilabiiiiy of the test compound can be generally ignored in foe in vitro system, the assay instead being focused primarily on the effect of the drug on tSm molecular target as may be manifest in an alteration of binding affinitybetween an AeiRlIa polypeptide and acts between an AetRlIb polypeptide and aetivin.

Merely to llldstrate, in ah exerngiafy screening assay of the present invention* the compound of interest is contacted with an isolated and purified ActRIIa polypeptide which is ordinarliy capable of binding to aetivin, To the mixture of the compound and ActRIIa polypeptide is then added a ebmppsitidh^ eohtaimpg an ActRIIa ligand. Detection and quantification oTAdfRtIa?a^iyid-iC»mplex^':p)^yid^:a..iJ^0n$ for determining the compound’s efficacy at InMbitmg (or potentiating) complex formation between the ActRIIa polypeptide and aetivin, The efficacy of foe compound can he assessed by genefotmg dose response carves from data obtained using various concentrations of the test compound. Moreover, a control iss^y can also be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified aetivin ls added to a composition eontamlpg: the AetRIJa polypeptide, and the ; formation of ActRIIa/activm complex is quantitated in the absence of the test compound. It will he Understood that, in general, the order-.hi which the reactants may be admixed can be varied, and can be admixed simultaneously; Moreover, in place of puri fied proteins* cellular extracts and lysates may be used to render a suitabie eoIT free assay system· Compounds that affect AetRlIb signaling may be identified m a similar manner using an AetRlIb polypeptide and an AetRlIb ligand.

Complex formation hetween the ActRII polypeptide and aetivin maybe detected by a variety of techniques. For instance, modulation of the formati on of complexes can he quantitated using, for example, deteetahly labeled proteins such as radiolabeled fe.g., UP, otJM), fluoreseently labeled fe.g., F1TC), or enzymatically labeled ActRliapr AetRlIb polypepide of aetivin, by immunoassay, or by chromatographic deteciiort- ln certain erhbodimenis, the present invention contemplates the use of Hyeresemtee polarization assays and duoreseence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between an ActRIl polypeptide and Its binding protein. Further, other modes of detection, such as those based on optical waveguides (FCT PufeHcatfon WO 96/26412 and II.S. Pat; No. 5,677,196), surface plasmon resonance (SPR), surfoce charge sensors, and surface force sensors, are compatible with many embodiments of the invention.

Momover, the present invention contemplates; the use of art interaction trap assay, also known as the-two hybrid assay,’" for i$^ifyipg· disrupt or potentiate interaction between an AetRII polypeptide and its binding protein- Bee for example,'U-S. Pat, No., 5,283317; Zervos et ah (1993) Cell 72:223-232; Madnrs et ai, (1993) :1 Biol Chern :2:68:12046-12054: Bartel et a!. (1993) Piotechniqnes 14'92:G“924; and iwabuchtet a|,;(1993:) Oncogene $: 1693 Ί 696), In a specific:embodiment, the: present invention contemplates the use of reverse;two hybrid systems to identity compounds (e.g„ small molecules or peptides) that dissociate interactions between an AetR 11 polypppfide-and its binding protein. See :fpr example, Vidal and Legram, (1999) Nucleic AeidaRes:27:919-29( Vidal mid Legram, (1999) Treft^Bioteehnbl 17374-81; and ITS, Pat, Nos, 5:,955,280; and ;5,965368,

In certain embodiments, the subject compounds are identified by their ability to imeraet with ah AetRll pr aetivin polypeptide of the invention, The inieractiombeiween the compound and the AetMIa, AetRilb, or activih; polypeptide may be covalent of nomcovai ent. For example, such interaction can be identified at the protein level using in vitro biochemical methods,, including phoiO~crossfinidng>: radiolabeled ligand, binding,: and affinity chromatography (Jakohy WB et a:L, 1974, Methods; in Engyrnolngy 46: 1). in certain esses, the compounds may he screened in a. mechanism based assay, such as an assay to detect compounds which bind to an actrvm or AetRII polypeptide- This may include a solid phase or fluid phase binding event. Alternati vely, the gene encoding an achyin or ActRII polypeptide can he transifeeted with a reporter system (e, g, , β-galactosidase, iueiferase, or green fluorescent pnotein) into a cell and screened against the library optionally by a high throughput screening or widx individual memhem of the library. Other mechanism based binding assays may be used, for example, binding assays which detect changes in free energy. Binding assays can he performed with the target fixed to a well, head or chip or captured:by an immohihxed antibody oryesolved by capillary electrophoresis, Thebound compounds may he detected usually using colorimetric or iuorescenee or surface plasmon resonance, 6. Exemplary therapeunc.Useg

In certain embodiments, activimActRIl antagonists (e,g,s AetRlhver ActRIlb polypeptides) of the present invention can be used to increase red blood cell levels i n mammals such asundents and primates, iia certain embodiments, thepsesent invention provides methods of treating or preventing anemia in an individual in need thereof by administering to tlriddlyti^e^et-a^utieglly effective amount of an aetivin-AeiRlIa antagonist, such as an AetRIia polypeptide, or a therapeutically effective amount of an achvin-AeiRilh antagonist, such as an AeiRIIb polypeptide, in certain embodiments» the present invention provides methods of promoting red blood cell formation in an individual by administering to the individual a therapeutically effective amount of aaactiyimAidEO antagonist, particularly an ActRIX polypeptide. These methods may M.i^c^ifor#«mp€pUctand:'|^ophyl3<^je'treamiep^if|hsthtnals»:'pnd:p&amp;ticulafiy humans.

As used herein, a therapeutic;that “prevents” a disorder or condition refers ip g compound that, in a statistical sample, reduces: the occurrence oTthe disorder or condition in the Peaied sample mlative to an untreated eonirol sa/nple, or delays the onset: or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. The term treating” as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it hasbeen 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

As shown herein, actiylmActRila antagonists and act i v i n A ct RI lb 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 cell pr hemoglobin levels, such as patients::having an anemia, and those that are at risk for developing undesirably low red blood cell or hemoglobin: levels, such as those patients that are about to undergo mator: surgery or other procedures: that may result in substantial blood loss. In one embodiment, a patient with adequate redblood cell levels is treated with an aettvimActRHa aniagonistto increase red blood cell levels, and then blood is drawn and stored: for later use in transfusions. In one embodiment, a patient with adequate red blood ceil levels is treated with: an aetsvm-AetROb antagonist to increase red blood cell levels, and then blood is drawn and stored for later use in transfusions.

ActiYtri-ActEll antagonists disclosed herein,and parltcuiatly ActRlia-fc and ActRllb proteins, may fee usedto increase red blood cel! levels in patients having an anemia. When observing hemoglobin levels in humans, a level of less than nomml for the appropriate age and gender category may he indicative p? anemia, although individual variations are taken into account. For example, a hemoglobin level of 12 g/df is generally considered the lower limit of normal in Ihe general adult population. Potential causes include Mood-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, myeiodyspiastic syndrome, rhenmafoM arthritis, bone marrow transplantation. Anemia may also be associated with the following conditions: solid· turners (e.g. breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (e,:g. elironic lymphocyte leukemia, nomBpdgfons and Hodgkins lymphomas); tumors of the hemaiopoieiesystem (eg, leukemia, niyelodyspiastie syndrome, multiple myeloma); radiation therapy; chemotherapy (e.g. platinum containing regimens); inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythemufoais (SLE), acute or chronic skin diseases (e.g. psoriasis), inflammatory bowel disease (eg. Crohn'S 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 trans&amp;sion of red blood cells is not possible due to patient alio- or auto-antibodies and/or for religious reasons (e.g. some Jehovah's Witnesses); infections (e,g. malaria, osteomyelitis); hemoglobmoptbics, iheluding, for example, sickle cell disease, thalassemias; drug use or abuse, e.g. alcohol misuse; pediatric patients with anemia from any cause to avoid transfusion; and elderly patients or patients with underlying cardiopulmonary disease with anemia who cannot receive transfusions due to concerns about circulatory overload.

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 about; 12,5 g/dl, and typically about 11.0 grill (see also Jacobs et at. (2000) Nephrol Dial Transplant 15, ! 5-19), although lower target levels may cause fewer cardiovascular side effects. Alternatively, hematoerh levels (percentage of the volume of a blood sample occupied by the cells) can be used as a measure for the condition of fed blood ceils. Hematocrit levels for healthy individuals range from 4! to 51% for adult males and from 35 to 45% for adult females. 'Target hematocrit levels ate usually around 30-33%. Moreover, hmi©giobiudiematoerit levds v8ry lroni person to person. Thus* optimally, the target hemoglobin/hematocrit level can he individualized for each patient.

The rapid effect on red blood cell levels of the aptivIn-AetRila antagonists disclosed herein indicate that these agents act by a different mechanism than Ipo, Accordingly, these antagonists may be useful for inereasing red blood cell and hetnoglohin levels in patients that do not respond well to Epo. For example, an actiyfa^AetRKa^^iiit pay he beneficial for a patient in which administering of a normal to increased (4300 Rj/kg(week} dose of Ipo does not result in the increase of hemoglobin level up to the target level. Patients with an inadequate Epo response are found for all types of anemia, hut higher numbers of nonresponders have been observed particularly frequently in patients with cancers and patients with end-stage renal disease. An inadequate response to Epo can 1m either constitpfive (i.e. observed upon the first treatment with Epo) or acquired fe,g> observed upon repeated treatment with Epo).

The activin-ActRlf antagonists 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 hemoglobin levels 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 headaches, influenza-like syndrome,,Obstruction of shunts, myocardial inferetians and cerebral convulsions due ip thrombosis, hypertensive encephalopathy* and red cell blood cell applasia (Sihgibarti, (1994) .1, Clin Inyestig ?2(syppi 6)* iS:36“S43;Tiprl et al, (2000) Nephrol Dial Transplant iSfsuppl 4), 51-56; Delanty et al, ¢1997} Neurology 49, 686-689; Bunn (2002) N Ehgl J Med 346(7), 52:2-523). 7. Pharmaceutical Compositions

In certain embodiments, activin-ActRli antagonists (e.g,, ActRifa and ActRIlb polypeptides) of the present invention are formulated with a pharmaceutically accepiabie carrier. For example, an ActRII polypeptide can be administered alone or as a component of a phapnaceutical formulation (therapeutic composition). The suyeei compounds may be formulated for administration m any convenient way for use in human or veterinary medicine.

In certain/^ljQdi.^^tSt.ili^iherai^ptit^e^od of the invention mefodes administering the composition systemlcally, or locally as an implant or device. When administered, the therapeutic composition for use in tfos invention i% ofcputpe, In a pyrogen-free^ physiologically acceptable form Therapeutically useful agents other than the aetivin-ActRII antagonists which may also optionally he included in the composition as described above, may he administered simultaneously or sequentially with the subject compounds {e.g,, ActRlla and AetRIlh polypeptides) in the methods of the in vention,

Typicallyi activin- ActRil antagonists will he administered parenteral! y.

Pharmaceutical compositions suitable for parenteral; administration may comprise one or more ActRl.i polypeptides in combination with one: or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted info sterile injecfahle solutions or dispersions just prior to use, which may contain antioxidants, buffers, haeteriostats, solutes which render the formulation isotonic with the blood of the intended recipient Or suspending or thiekemug agents. Examples of suitable1 aqueous and jmnaqueous carriem which may be employed in the pharmaceutical compositions of the inventiim include water, ethanol, polyols (such as. glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof vegetable oils, such as olive oil, and as ethyl oleate, Proper fluidity can he 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 surfactants.

Further, the composition may be eneapsulafod or itbeeted in a form for delivery to a target tissue site fe,g., hone marrow). In certain emhodlmeats, compositions ofthe present invention may include a matrix capable of delivering one of more therapeutic compounds fmg., AeiRlia or ActRllb polypeptides) to a target tissue sitefe,g., hone marrow), providing a structure for the developing tissue and optimal ly capable of being resorbed into foe body. For example, the matrix may provide slow release of the ActKfl polypeptides. Such matrices may be formed of materials presently in use for other i mplanted medical applications.

The cilice of .matrix material is based bn fefocompatibilit^» biodegradabihiy;, mechanical pmperiles, cosmetic appearance and interfeee properties, The particular applieation όίthe: sul^ept compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable arid eheniieally defined calcium sulfate, tricMcrumphosphate,hydroxyapatite, polylaetic acid and polysnhydrides. Other potential materials are biodegradable and biologically well defined,such as hone or dermal collagen. Further matrices are comprised of pure proteins or extrsceHidar matrix #mponents. Other potential matrices are noh-biodegmdable and chemically defined, such as sintered hydroxyapatite, bioglass, afuminates, or other cemmics. Matrices may he comprised of eombisiatiops of any of the above mentiosted types of material, such as-.polyiactic acid and hydroxyapatite or collagen and triealcimiiphosphate. The Moceramics may be altered in composition, such as in ealciam-aiuminate-phosphate arid processing to alter pore size, particle size, particle shape, and biodegradability.

In cert ain embodimeniss methods of the i nv ention can be admini stered for orally, e,g.5 in rite form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanih), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oildn-water or waterrinmi! liquid emulsion, or as an elixir of syrup, of as pastilles (using an inert base; such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washesand. the like, each containing a predetermined amount of an agent as an active ingredient. An agent may also be administered as a bolus, electuary or paste.

In solid dosage forms for Oral adnumsuation (capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present invention m ay be m ixed with one or mere pharmaceutically acceptable carriers, : such as sodium citrate or dicalcmm; phosphate, pud/or any of the following: (1) fillers or extendersjsuch as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2f binders, such as,, for example, catboxymethyiceltulose,:ai:ginates, gelatin, polyvinyl gyrrolidone, suefose, and/or acacia; (3) humeetanis, such as glycerol; ¢4) disintegratingi agentS, sneh as agar-agar, calcium carbonate, potato: or tapioca starch,:alginic acid, certain silicates, and sodium carbonate; (5) solution retardihg agents, such as paraffin; (6) absorption accelerators, sueh as quaternary ammonium compounds;::(7) wotting agents, such as, for example, cetyl alcohol and glycerol nmnostes?aiey{8) absorbents,, such askaoliu and bentonite ctaiyi (9) lnbricant% such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium: ferny! sulfate, and mixtures thereof; and (10) coloring agents, in the case of capsules* tablets and pills, the pharmaceutical compositions m ay also comprise buffering agents, Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high moieewlar x^eighf polyethylene glycols and the like, liquid dosage forms for oral adnainistratibd include pharmaeeufieaily aeeeptabie emulsions, mieroemnlsfons, solutions, suspensions, syrups, and elixirs, in addition to the active ingredient, the liquid dosage forms may :eontaln inert diluents commonly used in the art, such as water or other so:!veptsi:ap|nhi!izing: agents and emulsifiers, such as ethyl alcohol, isopropyl: alieohol, ethyl carbonate* ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol,: 1 ^-buiylene glycol, oils firi particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofury! alcohol, polyethylene glycols and fatty acid esters of sofbttam and mixtures timreof Besides inert diluents, the oral compositions canalso include adjuvants such as wetting: agents, einuisiiying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain snspending agents such as ethoxylated isosiearyl alcohols, polyoxyethylene sorbitol, and sorhitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mix tures 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 nucroorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, ehlorobutanol, phenol sorbic acid, and the like, it may also he desirable to include isotonieagents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaoeutieal farm may be brought about by the Inclusion of agents which delay absorption, such as aluminum mpnostearate and gelatin.

It is understood that the dosage regimen will be determined by shdaftending physician considering various factors which modify the action of the subject compounds of the invention (e.g,, Act&amp;IIa and AetRlIb polypeptides). The various factors include, hot are not limited to, the patient’s red Mood ceil coopt, hemoglobin level or other diagnostic assessments, the desired target red blood cell count, the paienfs ag®, sex>: and diet, the severity of any disease that may be contributing to a depressed red blood cell level, time of administration, and other elinicaiiactors. The addition of other known growth factors to the final composition may also affect the dosage. Progress can he monitored by periodic assessment of red blood cell and hemoglobin levels, as well as assessments of reticulocyte levels and other indicators of the hematopoietic: process,

Experiments with primates and: humans have demonstrated that effects of Aet!Mia-Fe on red blood cell level a 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 of200 ng/ml, 500 ngrinl, 1000 ng/ml or greater for a period of at!east 20 to 30 days may also fee used. Bone effects can be observed at serum; levels of about 200 ng/ml, witfe substantial effects beginning at about 1000 ng/fni 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 deli ver a serum concentration of between ahont 100 and 1000 ng/ml over a period of about 20 to 30 days. In humans, serum levels of200 nghnl may he achieved with a single dose of 0/1 m^kg or greater and semm fevelsofliOO ng/ml may be achieved with a single dose of 0.3 mg/kg or greater. The observed srsihp fealMife of the molecule is between about 20 and 30 days, substantially longer than roostFe fusion proteins, and thus a sustained effective serum-..level may fee achieved, for example, by dosing with about 0.05 to 0.3 mg/kg on a weekly or bi weekly basis, or higher doses may be used with longer intervals between dosings. For example, doses of 0T to 1 mg/kg might be used on a monthly or felmondtly basis, in certain embodiments, the present invention also provides gene therapy for the hi vivo production of ActPll polypeptides. Such therapy would achieve its therapeutic effect fey introduction of the ActRHa or ActFIfb polynucleotide sequences into ceils or tissues having the disorders as listed ahove, Delivery of AetRil polynucleotide sequences can he achieved usmg a recombinant expression vector such as a:chimeric virus ora colloidal dispersion system, Preferred-for therapeutic delivefy of AetRI! polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can he utilized for gene therapy as taught: .herein include· adesovirus, herpes virus, vaccinia, or an ENA virus such as a retrovirus.· The retroviral vector may be a. derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can he inserted include, hut are not limited to: boloney muri# leukemia virus :(MoMuLV)s Harvey murine sarcoma virus (HaMuSVf murine mammary tumor virus (MyMTV), and Rhus -Sarcoma Vims fESV), .A number of additional retroviral vectors can incorporate multiple genes,. All of these vectors can transfer or incorporate a gene lor a .selectable marker so that: transduced ceils can he identified and generated. Retroviral vectors cap he made target-specific by aiiaObmgjlfhr example, η sugar,. a glycolipid, or a protein. Preferred, targeting is accomplished by using an antibody. Those: of skill in the ait will recognize that speclie poiynueieotide sequences can be inserted into the retroviral genome or attached to;a viral envelope to allow target specific delivery of the retroviral vector eontaiuing the AotRU polynucleotide.

Alternatively, tissue culture cells can be direetiy transfected with plasmids encoding the retroviral stmetufal genes gag, pol and env, by conventional calcium phosphate transhmtidft. These cells are then transfected with the veetor plasmid containing the genes of interest. The resulting cells release fce mimviral vector into the culture medium.

Another targeted delivery system for ActRII poiynudeotides is a colloidal dispersion system^ Colloidal dispersion systetns include inacmmoleoule complexes, nanoeapsuies. mierosphemSi beads, and lipid-hased systems including oilbp-wMer emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes afe artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RhlA, DNA and intact virions ean be eueapsulated within the aqueous ihieriof and he delivered tb cells in a biologically active fermfsee e^g,, Fraley, et al„ Trends Bioehem, Soil, 6:77, 1981). Methods for efficient gene transfer using a liposome vehicle, are known in the arti see «.g., K4annino, et ai., Biotochniques, 1¾¾ The com position of the hposome is usually a eombinatioh of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. Theiphysical characteristics of liposomes depend ottpB, ionic; strength, and the presence of divalent cations.

Examples of lipids tweiui in liposome production indude phosphatidyl compounds, such as phosphatidylglyceroh phosphatkiyi choline, phosphatidylsetme, fthosphatidyfethanolaniine, sphingnlipids, cerehrosides. and gangliOSides. Illustrative phpsphohpKls include eggphosphatidylehoUne, ditmiotitoylphosphatidylciioline, and. distearaylphosphatidylchpline,; The targeting of liposomes is also possible based on,.tor example, org^-specificity*: eel!-specificity, and organeitempecificity and is known in tho art.

EXEMPUEIGATION

Thp invention now being generally1 described* it will he more readily understood hy reference to the following examples, whiohare included merely for purposes of illustration of; certain embodiments and embodiments of the present invention,; and are pot intended'to limi t the invention.

Example It ActRlia-Pc FasionProteins

Applicants constructed a soluble ActRliaiusionprotein that has the extracellular domain of human AetRlla fused to a human or mouse Eo topain with a minimal linker ih between. The constructs are referred to as AetRIla-hPe and AetRIfa'mFe, respectively.

AetRlIaHiFe is shown below as purified from GHGeell lines fSEQ IDMO: 7):

iEGRSETQEeLFFhiANWEKDRd:N<pGVEFGyGDK.DKRRHCFATWKNiSGSIEI¥KiQG

GWEPDfMGYDjRTDCVBRIO^SPEVYECCCEGNMGNEKFSYFPEMEYTQPTSNPYiPK ΡΕ0ΠΊΟβΤ^ΓθΡΡ€ΡΑΡΕΕΕΟΟΕΒνΕΕΐΡΡΚΕΚ:ΡΤΙ.Μ1ΕΒΤΡΕνΤϋ¥¥νΒν5ΗεΡΡΕνΚΡ

NWyVPGyE\rtdNAKTKPREEOYNSTYRVVSVi:rVI.liOPWi;NClKEVy.CKVSNKALP

VFlERTiiKAKGOFREPQVYIXPFSRBEMTKNOVSLTCLVEGFYFSDlAyEWiSNGOP SFGR;

The ActRiladiFeand Ac?R1Ia*mFc proteins were expressed in GHO cell lines. Three different leader sequences were considered; (i) Honey beemeilttin (HBMLfi MKFLVNYALVFMVVYISY1YA (SEQ IP NG; 8) (ii) Tissue Plasminogen Activator (TPA): MDAMKEGLGCVLLLCOAVF¥SP (SEQ ID NO: 9) (in) Native: NfGAAARLAFAVFEiSCSSGA (SEQ ID NO: 10).

The selected form employs the TP A leader and has dm following unprocessed amino acid sequence: MDAMKIlOLCCVLLLCGAVFVSPOAAiLGRSElQECLPFNANWEKOiTrNQTfjVEPOy

GDRDERRHOEATWKNlS0SIEiVNQGGWiT)DIN€TDRT0CVEKKDSPEVYFGO€EG

NMCNBKFSYFPEMBVTQPTSNPWPKPFiGGGTHTGEPGPAPELLGGPSYFLFFPKPK ΟΤΕΜί$1ΕΓΡΕ¥ΊΤ:ννν0ν$ΗΕΟΡΕνΚΕΝ\¥Υνί>ΩνΕνΗΝΑΚΤΕΡΚΕΕΟΥΝδ1'ΥΚ¥¥Ε

VI.T V LHQD WENOMEYKCKAfSNKA IT VPIERTISKAKGQPRBPQVTTLPPBREEMTKN

QVSLTGLVKGFYPSOlAVBWBSNGQFENNTKTTPPVLDSDGSFFLTSKITVDKSRWQ QGNVFSCSVMREALIINHYTQRSLSLSPGN. (SEQ 10 NO:i3)

This polypeptide is encoded by the following nucleic acid sequence:

ATGGATGCAATGAAGAGAGGGCTGl'GCTGTGTOCFGCrGCTGTGTGGAGCAGTCT

TCG'rTTCGCCCGGGGGCGCTATACTTGGTAGATeAGAAACTCAGGAGTGTGTrrT

TTTAATGGTAATTGGGAAAAAGAGAGAAGGAATCAAACTGGTGTTGAACCGTGTT

ATGGTGACAAAGATAAACGGGGGCATTGT1TYGCTACCTGGAAGA.ATATTTGTGQ

ITCCATI^GAATAGIGAAACAAGG'TOTTGGCTGGATGATATCIAACI'GCTATGAGA

GGAOFGATTGTGTAGAAAAAAAAGACAGCCCrGAAGTAl'ATTTGTGi'TCiCTGTGA

GGGOAATATOTGTAATGAAAAGTlTTYrrTATTTTGGGGAGATGGAAGTGACACAG

CCCAGTIOAAATGGAGTrAGAGGTAAGGCAGeCACCGGTGGTGGAAGTCACACAT

GGCGAGGGTGCCCAGCAGCTOAACTGGTGGGGGGAGGGTGAGTCTTCGTCTrCGC

GCGAAAACGGAAGGACACCCTGATGATC'rCCCGGACGGCTGAGGTGACATGCGTG

GTGGl'GGACGTGAGCeAGGAAGA€GCTGAGGTGAAGTTCAACTGGTAGGTGGAG

GGCGTGGAGGTGCATAATGGCAAGACAAAGCCGCGGGAGGAGCAGTAOAACAG oacgtacggtgtggtcagcgtgctcagcgtcgtggagcaggactggctgaatggg

AAGGAGTAGAAGTGCAAGGTCTCCAACAAAGCCGTCCCAGI'CGCCATCGAGAAA ACCATCTCGAAAGCGAAAGGGCAGCXGGGAGAAGCACaGGTGTACAGGCTGCCC CCATCCCGdGAGGAGATGACCAAGAAGGAGGTCAGGGTGAGCTGGCTGGTCAAA GGCTTCTATGCCAGCGACATGGCCGTGGAGTGGGAGAGGAA iOGGCAGCGGGAG AAGAACTAGAAGAGGACGGGTCCCGTGCTGGAGTCCGAGGGCIQG Π G1 I'GGTCT

AtAGCAAGC'reACCaTGGAC^AGkGdAOGTGOdAClCAaOeGA^CGTOTCl'CAt GClGGGTGAXGCATGAQGCTCTGGACAACCAfFrAGACGGAGAAGAGGCTGTCCCT GTCTCCGGGTAAATGAGAATTC (SEQ ID NO: 14)

Boil) AetEila-hFc and AetRlIa-mFc were remarkably 'amenable: to recombinant expression. As shown in figure R -the protein was purified as a singly well-defined peak of protein. N-terrninal sequencing revealed a stogie sequence of-dLGRSTQE (SEQ ID NO: 11). Purification could be achieved .by a series of column chromatography steps, including, for examples three or more of the following* in any order: protein A dutonmtography, Q sepbarose chromatography, phesrylsejfoaroseehromatography, axe exclusion lehromatography, and cation exclrange chromatography. The puri fication:co«ld be completed with viralfilirabon and buifor exchange, The AciRIladiFe protein was purified to : a purity of >98% as determined by size exclwsion chromatography and >95%. as determined by $I3S PAGE.

AciRlia-hFe and ActRl!a~mFc showed a high affinity for ligands, particularly activin A. GDF-!T or Activin A (GfeiA”) were immobilized on a Biaeore CM5 chip using standard amine coupling procedure, ActR 1 la-hFc and AetRiia-mEe proteins were loaded onto the system, and binding was measured. AetRIIa-hFe hound to activin with a dissociation constant (Kp) oiSxlO*2, and thepxoiein hound to GDFll with a R® of 9.96x10s. See figure 2; AetRIla-mFc behaved similarly,

The AGElla-hFc was very stable in pharmacokinetic studies. RaG were dosed with 1 rng/kg,'% mg/kg or 10 mg/kg of AelRlla-hFe protein and plasma levels of the protein were measured at 24,48, 7% 144 and 108 hours. In a separate study* rats were dosed at 1 mg/kg* 10 mg/kg or 30 mg/kg, IP rats, AoiRIla-hFe had m 11-14 day serum ha!f life and circulating levels of the drug: were quite high after two weeks (:11 gg/ml, |iO pg/ml or 304 gg/nfi for initial administrations of 1 mg/kg, 10 rng'xgor 30 mg/kg, respectively.) In eyuomolgus monkeys, the plasma half life: was substantially greater than 14 days and: circulating levels of the drug were;25: qg/ml, 304 gg/ml or 1440)gg/mi for initial admlnritratlohs of I?mg/kg, 10 mg/kg of 30 rng/kg, respectively.

The study employed four groups of five male arid five female cynomo I gus moneys eaeh, with threepet sex per group scheduled fer termination on Day/SSfoaocl two per sex per group scheduled for teirniMiion on Day S?. Each animal was administered the vehicle (Group 1} or ActEIia»Fe at doses of li: 10, or; |0 nig/kg (Groups 2» 3 and 4 respectively) via intravenous (IV) injection rmlDeys 1,8, tfeund 22. llies-dose' yolu^e-w^:matp|aiaie>d at 3 mL/kg. Various measures of red bl ood cell levels were assessed two; days prior to the first administration and af days IS, 29 and 57'ffprthe remaining two animals) after dm first administration, lire AeiRila~hFc causes statistically significant Increases in mean red blood cell parameters (red Mopdl cell count [RB€], hemoglobin: [HGB], and hematocrit 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.

Statistical significance! was calculated for each treatment group relative to the mean for the treatment group at baseline.

Motably, the increases In red blood coll counts and hemoglobin levels are roughly equivalent In magnitude to effects ported with erythropoietin. The onset of these effects is more rapid with ActKIla-Fe than with erythropoietin.

Similar results were observed with rats and mice.

Example 3; ActRIfe-hFe Increases Red Blood CeflJfeyel^^

The ActMila-hFc fusion protein described in Example 1 was adininistered to human patients in a randomized, double-blind, placebo-coTUtolled study that was conducted to evaluate, primarily, the safety of the protein in healthy, postmenopausal wonfem Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ActRlla-liFc or placebo (5 active; I placebo). Dose levels ranged from ¢),03 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.3 mg/kg subcutaneously pG):, :All sul^ects: were followed for 120 days. In addition to pharmacokinetic (PK) analyses, the biologic: activity of ActRlIa-hFc was also assessed by measurement of biochemical markers of bone fonuafipn and resorption, and F:SM levels.

To look for potential changes, hemoglobin and RBC numbers were exam ine&amp;fn detail for all subjects over the course of tie study and compared to the: baseline levels. Platelet counts were compared; over the same time as; the control I'here were fio clinically significant changes from the baseline values over time for the platelet counts. PIC analysis of ActROa-liFc displayed a linear profile with dose, and a mean half-life of approximately 25-32 days, Theareamnder-curve (AUC) ibrAetRIla-hFe was linearly relatcdfo dose, and the absorption after SC dosing was essentiailyeompleie (see Figures 7 and 8), These data indicate that SC; is a desirable approach to dosing because it provides eqidvalent hioavailsbility 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). AciRIIa-hFe caused a rapid„ sustained dpse-dependent increase: ih:serum levels ofheme-specihc alkalinephosphatase (BAF),, which is a m arker for anabolic hone growth, and a dose-dependent decrease in C-terminai type 1 collagen teiopeptidc and tartrate-resistant acid phosphatase 5b levels, which are markers:fpr hone:resorption, Other maricers, such as PINP showed mconclhsive results. BAP levels showed near saturating effects at the highest dosage of drogvindieating: that half-maximal effects on this anabobe horie hlomarker could be achieved at a dosage of 0,3 rng/kg, with increases ranging up to 3 rng/kg. Caleulated as a relationship of pharmacodynamic effectdp- ABC for drug, the BCSCf is 51,4t>5 fday^ugrmiy See Figure 9, These bone bi^9markerhhe^ies:'W<^e.''St^Sh'ed^P;a^pro>xtmayiy 120 days at the highest dose levels tested. There was also a dose-dependent decrease in serum FSM levels consistent with inhibition of activin.

Overall, there was a very small non-dmg related reduction in hemoglobin over the fhst week of the study probably related to study phiebotoiny in the 0.01 and 0.03 mg/kg groups whether given IV or SC, The0;1 mg/kgSO and IVhemoglobin results were stable or showed modest increases by Day g-15. At the 9.3 mg^kg iy dose level there was a clear inereasein BOB levels seen as earl y as ©ay 2 and often peaking at Day 15-29 that was not seen in the placebo subjects. At this point in the study, this change has not reached Statistical signiicance.

Overall, AetRlIa-hPc showed a dose-dependent eifeet on red blood cell counts and reticulocyte counts. Fora summary of hematological changes, see Figures 10-13.

Example 4: Alternative ActROa-Pc Proteins A variety of AGRlla variants that may be used according to the methods described herein are: described in the: international Patent Applteation published as WO2OO6/012627 (see&amp;g., pp, 53-58¾ m<m|p^rat^'b^in'%fdfer^eedri its entirety. An alternative construct may have a deletion of theG-iennmal tad (the fitlM 15 amino aeid|ofthe extiaceitelar domain of AetRlla. The sequence for such: a. construct is presented below (Fe portion uuderlined)pEQ ID NO: 12):

ILGilSETQECLFFNANWEKDRTNQIOVBPCYGDKDMMRHOFAXWICMiSGSlEIVKQO

OWmDTNGYPRTDCVERlCDSPEVYFCCCEGNMCNERFSYFPEMTOGOMiYlFPCPA PELLGdPSVFLFFFKPK.DTLMiEim>EVTCVVYDVSHEPP;EYK.FNW¥VDG¥EVHNAK: TRFREEOYNSTYRVVSMidrYFJfOPWLNGKEYKC^YSNRALPYPmKTliiCAKGOPRE:

FOVYTLPFSREEMTK'NOVlErGLVKGFYPSPlAYEWESNGOPEhiNYKTTFFVIJDSPG

SFFi^YKLTViDYSRWOOGNVFSCSVMtlEALriNHYTOKSLSLSPGR

Example s: ActRllb-Fc Fusion Proteins

Applicants constructed a soluble ActRlib fusion protein that has the extracellular domain of human AetRiife fused to a human Fe domain Aeo-erystal structure of A etivin and extracellular ActRlib did not show any role for the Rnal fG4ennin§l) 15 amino acids (referred to as the -halF hereisYof the extraeeilular domain in ligand binding. This sequence foiled to resolve on the crystal slmmum, suggesting foat these residnes are present in a flexible loop that did not pack unifonnly in the crystal. Thompson et ah EM80 J, 2003 Apr ];22(7): 1555-66, This sequence is also poorly conserved between AetRlIb and AetRlla. Accordingly, these residues were Omitted in the basic, or background. ActRllb-Fc fusion construct Additionally, position 64 in the background form is occupied by an qlanine, which is generally considered the “wild type” form, although a A64R allele occurs naturally. Thus, the backgrotmd ActRllb-Fc fusion has the sequence {Fe portion underlined)(SE0 ID NO:20); SGROEAfTRECiYY^AI^WELE^THQS^ERCB^BQSliEl^HGYASW^iN^^OTlELVK KCC WLDOFN C Y PRD EC V ATE BN PO¥Y FCCCEOHFCM ER FT!i LP £ AGG DTE TCP PCP APELEGGPSVFEgPPKPKm:0^1SRTPEVTieV¥VPVSHfeDPEV^gNWYVDe^VMHA K^RPREEOY^STYRVVSYCTYLeDDWLMGKEYKCKVSMKALFVPIEKTiSRAIfteOF R EPOV YTEP P S RBEMTR.N OVS ^ΙΥ^ΕΥΙ^βΡΥ PSOIA YE W ESNGQPEHNYRTTPPy LDS :Βα5ΡΕΕ¥8ΚΕΤν0ΚΕ.Κ^νΡΟΟΝ:ΥΡ3€!8.ΥΜί1ΕΑ:ΕΗΜΗΥΤΟΚ8Ε51,^ΡΟΚ·

Surprisingly, the CAonmodl tail was found to enhance aeriyin and GDFG1 binding, thus a presforeed version of ADRHh-Fchas a sequence (Fe portion «oderiined){SH<| ID »21):

SGRGEAETRECIYY^ANWItERTNQSGLERCEGEQPKRLHCYASWAMSSGTIELVK

KGCWhDDFNCYDR^ECYATEENPQYYFCCGEGNPChlERrrHLPEAGGFE^TYEPPP ΤΑΡΤΟβΟΤΗΤ€ΡΡ€ΡΑΡΕΙ,ΕαθΡ8ΥΡ;ΕΕΡΡΚΡΚΡΤΙΜ15ΚΤΡΕνΤ(:νννΐ>νΕΗΕΡΡΕν

KFNWY¥DG¥E¥MNAKTKPREE0YNSTYR¥¥SYLT¥LiK)DWENGREYRCR¥SMKA

LPVPmRTlSI£AM.GOPRBPOVVTLPPSR£EMTK^GYSETCLVKGEYPSDIAVEWESHG

0ΡΕΝΝΥμ:ΓΤΡΡΥΕΡ3ΡΟ8ΡΡΕΥΕΚΕΤΥΡΚ3 rwogqpiyfscs vmhealhnh YTOKSL

StSPGK. A variety nfAetRIIb a variants that may he used according to the methods described herein are described in the: international. Patent Appliparinn p«Wished as WD2Q06/OI262? (see e.g., pp, 59-60), incorporated herein by reference in its entirety.

Example 6; ActRlIb-hFc Stimulates Erythropoiesis in Non-Human Primates

ActRBh-hFe (IgGI) was administered once a week tor 1-month to male and female eynomolgus monkeys by swEcntaheons hyeetion. Fortyreight cyndmolgtjs monkeys (24/sex) were assigned to one of fear treatment groups(6 animats/sex/group) and were administered subcutaneous infections of Cither Vehicle or AetR!ib-hFe at 3, 1.0, or 30 mg/kg once weekly for 4 weeks (iota! of 5 doses)· Parameters evaluated included general cl in ieai pathology (hematology, clinical chemistry, coagnlation, and urinalysis), AetRllb-hFc caused statisheally significant elevated mean absolute reticulocyte values by day 15 In treated animals. By day 36, AetRllb-hFc caused' sevefal hematoiogleai changes, including elevated mean absolute reticulocyte and red blood eell distribtttion width values and lower mean corpuscular hemoglobin concentration. All treated groups and both sexes were affected.

These ©fleets are consistent with a positive effect of AetRIlh-hfc oo the T^iease oFiimBatnre reticulocytes from the bone marrow. This effect was reversed after dmg was washed oat of the treated animals (by study day 5b), Accordingly, we conclude that ActRIlb-hFe stimulates erythrOpoiesis.

IITORPORATIQN BY REFERBMCE

All publications and patents anentioned herein are hereby ineerpomted by reference in their entirety asif each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject matter have: been discussed, the above speet.fieatidn·'is illustrative and;not restrictive. Mnhy variations will become apparent to those skilled in the art upon review of this: specification and the Claims below. The full scope ofthe invention should he determined by reference to the claims,: along with: their full scope of equivalents, and theepeei'Rcationv along with such: variations.

Claims (22)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:-

   1. A method for promoting erythropoiesis in a patient, the method comprising administering to a patient in need thereof an effective amount of an ActRII polypeptide selected from: a) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2; b) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NOG; c) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; d) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17; e) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:7; f) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 12; g) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:20; and h) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:21.
2. 2. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NOG.
3. 3. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NOG.
4. 4. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NOG.
5. 5. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NOG.
6. 6. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16.
7. 7. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 16.
8. 8. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17. 9 The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 17.
9. 10. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:20.
10. 11. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:20.
11. 12. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:21.
12. 13. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:21.
13. 14. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:7.
14. 15. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7.
15. 16. The method of either one of claims 14 or 15, wherein the N-terminus of the polypeptide is ILGRSETQE (SEQ ID NO: 11).
16. 17. The method of any one of claims 1-16, wherein the polypeptide has one or more of the following characteristics: i) binds to an ActRII ligand with a Kd of at least 10' M; and ii) inhibits ActRII signaling in a cell.
17. 18. The method of any one of claims 1-17, wherein the polypeptide binds to activin A.
18. 19. The method of any one of claims 1-18, wherein the polypeptide is a fusion protein comprising, in addition to an ActRII polypeptide domain, one or more polypeptide portions that enhance one or more of in vivo stability, in vivo halflife, uptake, tissue localization or distribution, formation of protein complexes, and/or purification.
19. 20. The method of claim 19, wherein the fusion protein comprises a polypeptide portion selected from: an immunoglobulin Fc domain and a serum albumin.
20. 21. The method of claim 19, wherein the fusion protein comprises a constant region from an IgGl heavy chain.
21. 22. The method of any one of claims 1-21, wherein said polypeptide comprises one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety.
22. 23. Use of an ActRII polypeptide selected from: a) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2; b) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NOG; c) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; d) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17; e) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:7; f) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 12; g) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:20; and h) a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:21, for the manufacture of a medicament for promoting erythropoiesis.