AU2015202035A1 - Use of GDF traps to increase 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 primates, and particularly in humans.

Description

USE OF GDF TRAPS TO INCREASE RED BLOOD CELL LEVELS RELATED APPLICATIONS The present application is a divisional application of Australian Application No. 2009282441, which is incorporated in its entirety herein by reference. 5 This application claims the benefit of U.S. Provisional Application Serial No. 61/189,094, filed August 14, 2008. All the teachings of the above-referenced application are incorperated herein by reference. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be 0 considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. The mature red blood cell, or erythrocyte, is responsible for oxygen transport in the circulatory systems of vertebrates. Red blood cells carry high concentrations of hemoglobin, a protein that binds oxygen in the lungs at relatively high partial pressure of oxygen (pO2) 5 and delivers oxygen to areas of the body with a relatively low PO2. Mature red blood cells are produced from pluripotent hematopoietic stem cells in a process termed erythropoiesis. Postnatal 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 20 conditions, red blood cells are produced at a rate that maintains a constant red cell mass in the body, and production may increase or decrease in response to various stimuli, including 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 25 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 reticulocyte:erythrocyte ratio, in the blood is indicative of increased red blood cell production rates. Erythropoietin (Epo) is widely recognized as the most significant positive regulator of 30 erythropoiesis in post-natal vertebrates. Epo regulates the compensatory erythropoietic 1 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. la Various Iorms of recombinant Epo are used by physicians to increase red blood cell levels in a variety ofncinial setings and particularly for the treatment of anemia. Anemia 1i 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, Morecommonly, anemia is secondary to diseases of other systems (Weatherall & Provan (2000) Lancet 355, 1169-i 175). Anemia may result from a reduced rate of production or increased rate of destruction of red blood ceils 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, chemotherapy treatment, nyelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation, Treatment with Epo typically causes a rise in heroglobins by about 1 -3 gdL 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 iirovements n quality of life and prolonged survival Epo is not uniformly effective, and many individuals are refractory to even high doses (-orI et al (2000) Nephrol Dial Transplant 15, 43-50), Over 50% of patents with cancer have an inadequate response to Epo, approximately 10% with end-stage renat disease are hyporesponsive (GIaspy et al. (1997) J Clin Oncol 15, 1 218 -1 234; Demetri ei at (1998) J Clin Oncol 16, 3412-3425 and less than 10% with myelodysplastic syndrome respond favorably (Estey (2003) Curr Opin ematol 10, 60--67), Several factors, including inflamination, iron and vitamin deficiency, inadequate dialysis, aluminuntoxicity and hvperpratroi may predict a por rapeutic rose, the molecular mechanisms of resistance to Epo are as yet unclear, Thus, it i an obiet of the present disclosure to provide alterative compositions and methods fr incasing rgr blood cell levels in patients SUMMARY OF THE INVENTION In park, the disclosure deminstrates that GDF Traps may be used to increase red blood cell and hemoglobin levels, Variant ActRilB polypeptides having a significantly decreased affinity for activrin (esg. activin A and/or activin B) relative to other ActR 11B ligands such as GDF I I and/or miostatin. are referred to as GDF Traps. ActR lB vanants described herein 2 areODE Traps unless otherwise stated. in particular, the disclosure demonstrates that a ODF Trap which is a solUble form of AcMRUB polypeptide having an acidic residue at position 79 of SEQID NO: 1, when administered in vivo, increases red blood cllevel in the blood. Therefore in certain eribodiments, the disdosure provides methods for using GDF Traps to increase red blood cel and hemolobin Jevels in patients and to treat disorders associated with low red blood cell or hemoglobin levels in patients in need thereof As described in US Patent Application No. 12/012,652 incorporated by reference herein GDF Traps can be used to increase muscle mass and decrease Pat mass in certain aspects, the present disclosure provides DF Traps that are vacant ActRIl1 polypeptides, including ActRlB polypeptides having amino- and carboxy-terminal truncations and sequence alterations. Optionally, GDF aps of the invention may he designed to preferentially antagonize one or more ligands of ActRIIB receptorssuch as GDEF (also called rmyostairn) ODF 1, Nodal, and BMP7 (also called OPl), Examples of ODE Traps include a set of variants derived from ActRi13 that have greatly diminished affinity for activin, These variants exhibit desirable effects on red blood cells while reducing effects on other tissues. Examples of such variants include those having an acidic amino acid (eg., aspartic acid, D, or glutamic acid, E) at the position corresponding to position 79 of SEQ ID NOL In certain embodiments, the ODE Trap polypeptide comprises an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 7, 26, 28, 29, 37 or 38, and polypeptides that are at least 80%, 85%, 90% 95% 97%, 98%, or 99% identical to any of the foregoing In certain aspects, the disclosure provides phannaceutica preparations comprising a GDP Trap that binds to an ActRlB 1 ligand such as ODF, GDF I activin (e g activin B) BMP7 or nodal, and a phamaceutically acceptable cater Optionally, the GDF Trap binds to an ActR ilB hgand with a Cd less than 10 micromofar, less than I nicromolarz less than 100 nanomolar ess than 10 nanonolar, or less than I nanomolar. Optionally the GDF Trap inhibits ActRlBf signaling, such as intracellular signal transduction events triggered by an ActHE ligand A GDF Trap for use in such a preparation may be any of those disclosed herein, including, for exmape GDF Taps baingan amino acid sequence selected front SEQ ID NOs 2, 3, 7, 11, 26, 28. 29, 32, 37 8 or 40. or ODE Traps having an amino acid sequence that is at least 80%, 85% 90%, 95% 9% or 99% identical to an amino acid sequence selected from SEQ i NOs 2 3, 7, 11, 26, 28, 29 32, 37, 38 or 40, or GDF Traps 3 having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amno acid sequence selected from SEQ ID NOs: 2 , 7, 28, 29, 32, 37, 38 or 40 wherein the position corresponding to 179 in SEQ ID NO: I is an acidic amino acid. A preferred GDE Trap for use in such a preparation consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 26. A GDF Trap may include a functional fragment of a natural ActR 1i B pol ypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID N.s: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38 or 40 or a sequence of SEQ ID NO: 2, lacking the C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at the N-terminus, A preferred polypeptide will comprise a truncation relative to SEQ ID NO: 2 or 40 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus. A GDF Trap may include one or more alterations in the amino acid sequence of an ActR1iB polypeptide (e.g., in the ligand-binding domain) relative to a naturally occurring ActIRUB polypeptide. The alteration in the amino acid sequence may, for example, alter glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRIIB polypeptide. A GDF Trap may be a fusion protein that has, as one domain, an ActRli3 polypeptide (e.g, a ligand-binding domain of an ActR I I with one or more sequence variations) and one or more additional domains that provide a desirable property, such as improved pharnmacokinetics, easier purification, targeting to particular tissues, etc. For example, a domain of a fusion protein may enhance one or more of in vivo stabi lity, in vivo half life, uptake/administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification. GDF Trap fusion proteins may include an immunog1otulin Fe domain (wild-type or mutant) or a serum albumin. In certain embodiments, a GDF Trap fusion comprises a relatively unstructured linker positioned between the Fe domain and the extracellular ActRUlB domain. This unstructured tinker may correspond to the roughly 15 amino acid unstructured region at the C-terminal end of the extracellular domain of ActRilB (the "tail"), or it may be an artificial sequence of between 3 and 5, 15, 20. 30, 50 or more amino acids that are relatively free of secondary structure. A tinker may be rich in glyci ne and proline residues and may, for example, contain repeating sequences of threonine/serine and e ., TG 4 (SEQ ID NO: 13) or SG4 (SEQ ID NO: 14) singlets or repeats) or a series of three glycines, A fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a 4 GST fusion. in certain embodiments a GDF Trap fusion comprises a leader sequence, The leader sequence may be a native ActRIB leader sequence or a heterologous leader sequence, In certain embodiments. the leader sequence is a Tissue Plasminogen Activator ('TPA) leader sequence In an embodiment, a GDF Trap fusion protein comprises an amino acid sequence as set forth in the formua A -. The B portion is an N- and Cterminally truncated ActRilB polypeptide consisting of the amino acid sequence corresponding to amino acids 25131 of SEQ ID NO. 2 or 40. The A and C portions may be independently zero, one or more than one amino acids, and both A and C portions are heterologous to 1- The A and/or C portions may be attached to the B portion via anker sequence. Optionally, a GDF Trap includes a variant ActRB 13 polypeptide having one or more modified amno acid residues selected from: a glyosylaited amino acid, a PE~ylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino a-cid conjiugated to a lipid, moiety, and an amino acid conjiaated to an organaic derivatizing agent. A pharmaceutical preparation may also include one rn nmore additional compounds such as a compound that is used to treat an ActRlIB-associated disorder, Pretrabiy, a pharmaceutical preparation is substantia ly pyrogen free. In general it is preferable that a GDE Trap be expressed hn a mamnmalian cell line that mnediatcs suitably natural glycosylation of the GDP Trap so as to diminish the likelihood of an unfavorable imune response in a patient. H--lunan and C-1 cell lines have been used successfully, and it is expected that other common mammalian expression vectors will be useful. In certain aspects, the disclosure provides packaged pharmaceuticals comprising a pharmaceutical preparation described herein and labeled fob use in increasing red blood cell levels in a human. In certain aspects, the disclosure provides GDF Traps which are soluble ActRi1B 5 polypeptides comprising an altered ligand-binding (eg, ODFS-binding) domata. GDFP raps with altered igand-binding domains may comprise for example one or more mutations at amino acid residues such as E37. E39, R40, KS5 .R6 60,A64, K74, W78,9 DS, F82 and P101 of human ActRilB (numbering is relative to SEQ ID NO: I), Optionally, the altered Ii ga nd-bi ibing domain can have increased selectiy for a ligand such as GDF8/GOF relative to a wild-type Iigand-binding domain of an AtRIl1B receptor, To illustrate these mutations are demonstrated herein to increase the selectivity of the altered ligand-binding doman %o GD Il (and therefore, presumably, GDF8) over activin: K474Y, IK74F 74n D9, L?9E. and D801 The following ntadons have the reverse effect, increasing the ratio of activin binding over GDF II: DS4A, K55A. L9A and F82A. The overall (ODF 1 1 and activin) binding actiity can he increased by inclusion of the "tail" region or, result an unstructured linker region, and also by use of a K74A mutation. Other mutations that caused an overall decrease in ligand binding affinity include: R40A, E37A, R56A, 4178A, D8OK, DSOR DS0k DS0Q, D80F DF, O)M and D80N. Mutations may be combined to achieve desired effects. For example, many of the mutations that affect the ratio of GDFl I Activin binding have an overall negative effect on Iigand binding, and therefore these may be combined with stationss iat generally increase ligand binding to produce an improvd bindng protein selectivity 61an exemplary embodi meant, a GDF Trap is an ActilB polypeptide comprising an [79D or [9E mutation, optioailly in combination with additional am no acid substitutions additions or deletions Optionally, a GDF Trap comprising an altered ligand-inding domain has a ratio of Kc for activin binding to Kj for GDFS binding that is at least 2, 5, 10, or even 100 fold greater relative to the ratio for the wikdtype ligandbinding domain. Optionally, the GDF Trap comprising an altered ligand-binding domain has a ratio of 1 csu for inhibiting activin to ICs for inhibiting ODF8/GDE I I that is at least 2, 5, 10, or even 1010 told greater relative to the wild-type ActR H B ligand -binding domain. Optionaly, the (GDF 'rap comprising an alered ligand-binding domain inhibits GDFS/GDPI wifi in IC o at least 2, 5, 10. or even 100 times less than the s for inhibitng activin Ibese GD 'raps can be nusion proteins that include an immunoglobulin Fe domain(either wild type or mutant) In certain cases, the subject soluble GDF [aps are antagonists (inhibitors)of cDF8 and/or GD11 Other ODFTraps are contemplated, such as the flowing. A GDF 'Frap fusion protein comprising a portion derived from the ActR I1IB sequence of SEQ ID NO: I or 39 and a second polypeptide portion, wherein the portion derived fiom ActR 1B eorresponds to a sequence beginning at any of arino acids 2-29 of SEQ U) NO: I or 39 (opnionaily beginning at 2225 of SEQ ID NO: I or 39) and ending at any of amino acids 109- 134 of SEQ ID NO: I or 39, and wherein the GDF Trap fusion protein inhibits signaling by activin, myostatin and/or GDl 1I in a cell-based assay The GDE Trap fision protein above, wherein the portion derived from ActRI1B corresponds to a sequence beginning at any of amino acids 20-29 of SEQ ID NO: I or 39 (optionally beginning at 2225 of SEQ I1D NO or 39) and ending at any of amino acids 109-133 of SEQ ID NO: I or 39 The GDF Trap fusion protein 6 above, wherein the portion derived from ActRUlB corresponds to a sequence beginning at any of amiino acids 20-24 of SEQ ID NO: I or 39 (optionady beginning at 22-25 of SEQ ID NO: I or 39) and ending at any of amino acids 109-133 of SEQ ID NO: or 3 The GDF Trap fusion protein above, wherein the portion derived fron AcRIIB corresponds to a sequence beginning at any of amino adds 2 [24 of SEQ ID NO: 1or 39 and ending at any of ammo acids 109-134 of SEQ ID NO: I or 39. The GDF Trap fusion protein above, wherein the portion derived from ActRii corresponds to a sequence beginning at any of amino acids 20 24 of SEQ ID NO I or 39 and ending at any of amino acids I 18133 of SEQ I) NO: I or 39. The GDF Trap fusion protein above, wherein the portioni derived from ActRilB corresponds to a sequencebeginning at any of amino acids 21 -24 of SEQ ID NO I or 39 and ending at any of amino acids I 18-. 134 of SEQ iD) N 0: 1 or 39. The GDE Trap fusion protein above, wherein the portion derived from AcIRiIB corresponds to a sequence beginning at any of amino acids 20-24 of SEQ ID NO: I or 39 and ending at any of amino acids 128W133 of SEQ 1D NO: I or 39, The ODE Trap fusion protein above, werein the portion derived from ActRIIB corresponds to a sequence beginning at any of amino acids 20 24 of SEQ ID NO: I or 39 and ending at any of amiuno acids 128-133 of SEQ ID NO: I or 39, The GDF Trap fusion protein above, wherein the portion derived forn ActRiIB corresponds to a sequence beginning at any of amino acids 21-29 of SEQ ID NO: I or 39 and ending at any of amino acids II-A134 of SEQ ID NO: I or 39. The ODF Trap fusion protein above. wherein the portion derived from AtR 11B corresponds to a sequence beginning at any of arnino acids 20429 of SEQ ID NO: I or 39 and ending at any of amino acids 118-133 of SEQ ID NO: I or 39. The DFT ap fusion protein above, wherein the portion derived from A ctR 111 corresponds to a sequence beginning at any of amino acids 21 29 of SEQ ID NO: I or 39 and ending at any ofarmino acids 128- 134 of SEQ ID NO: I or 39. The GDF Trap fusion protein above, wherein the portion delved from ActRliB corresponds to a sequence beginning at any of amino acids 20-29 of SEQ ID NO: I and ending at any of amino acids 128- 33 of SEQ 10 NO: i or 39. Surprisingly constructs beginning at 22-25 of SEQ ID NO; I or 39 have activity levels greater than proteins having the full extracelIular domain of human ActR11B3 In a preferred embodiment the DF Trap fusion protein comprises. consists essentially oh or consists of, an amino acid sequence beginning at amino acid position 25 of SEQ0 I NOI or 39 and ending at amino acid position 131 of SEQ ID NO: 1 or 39, In another preferred embodiments, the GDF Trap polypeptide consists of or consists essentially of, the amnio acid sequence of SEQ ID NO: 7, 7 26S 28 9, 32, 37 or 38 Any of the above GDF Trap fusion proteins may be produced as a homodimer Any of the above GDF Trap fusion proteins may have a heterologous portion that comprises a constant region from an IgG heavy chain, such as an Fe domain. Any of the above GDF Trap fusion proteins may comprise an acidic ainio acid at the position corresponding to position 79 of SEQ ID NO I optionally in combination with one or more additional amino acid substitutions, deletions or insertions relative to SEQ ID NO> t Other GDF Trap proteins are contemplated, such as the following. A GDEFJrap protein comprising an amino acid sequence that is at least 80% to the" s ne of amino acids 29009 of SEQ ID NO: or 39, wherein the position corresponding to 64 of SEQ ID NO: I is an R or K and wherein the GDP Trap protein inhibits signaling by activin, myostatin and/or GDF I in a cell-based assay. The GDF Trap protein abovewherein at least one alteration with respect to the sequence of SEQ IL NO: I or 39 is posiioned outside of the ligand binding pocket, The OD Trap protein aboevherein at least one alteration with respect to the sequence of SEQ ID NO: I or 39 is a conservative alteration positioned within the ligand binding pocket. The GDF Trap protein above, wherein at least one alteration with respect to the sequence of SEQ ID NO: I or 39 is an alteration at one or more Positions selected from the group consisting of K, R407 Q3 K55, F82 and L79. The GDF Trap protein above, wherein the protein comprises at least one N-X-S/T sequence at a position other than an endogenous N-X-S/T sequence of ActRI 11B, and at a position outside of the ligand binding pocket, Other GDF Traps are contemplated; such as the following, A ODE Trap protein comprisinAg anamino acid sequence that is at least 80% identical to the sequence of amino acids 29I09 of SEQ ID NO I or 39 and wherein the protein comprises at least one -NSIT sequence at a position other than an endogenous lN-X-ST sequence of ActRUIB. and at a posdion outside of he ligand binding pocket,. The ODE Trap above. wherein the GDF Trap protein comprises an N at the position corresponding to position 24 uf SEQ ID NO: I or 39 and an S or T at the position corresponding to position 26 of SEQ ID NO: I or 9 and whereinthe GDE Trap inhibits signaling by action, nmyostatin and/or ODE I in a cell-based assay The GDF Trap above, wherein the GD.P Trap protein comprises an T or K at the Position corresponding to position 64 of S EQ ID NO: i or 39. The CDF Trap above, wherein the ActRlb protein comprises a D or E at the position corresponding to position 79 of SEQ ID NO: I or 39 and wherein he ODE Trap inhibits signing by activin, myostatin and/or 8 GDOFi I in a celhbased assay, The GDF Trap above, wherein at least one alteration with respect to the sequence of SEQ ID NO. I or 39 is a conservative alteration positioned within the ligand binding pocket- The GDF Trap above, wherein at least one alteration with respect to the sequence of SEQ ID NO: i or 39 is an alteration at one or more positions selected from the group consisting of K74, R4. 053, K55; F82 and L79. The GDF Trap above, wherein the protein is a fusion protein further comprising a heterologous portion, Any of the above GDF Trap fusion proteins niay be produced as a homodinier Any of the above GDF Trap fu rteins May have a heterologous Portion that comprises consta region fom a IgO heavy chain, such as an c domain In certain aspects, the disclosure provides nucleic acids encoding a GDF Trap polypTeptide, An isolated poyrmueleotide nay comprise a coding sequence for a soluble GDF Trap polypseptide, such as described above, For example, an isolated nuclei acid may include a sequence coding for a GDF Trap comprising an extracelular domain (e g ligand binding domain) of an ActRII polypeptide having one or more sequence variations and a sequence that would code for part or al of' the transnmembrane domain and/or the cytoplasmic domain of an ActR.lIB polypeptide, but for a stop codon positioned within the transmemrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transnmebrane domain or cytoplasmic doman For example, an isolated polynueleotide coding for a GDF Trap may comprise a full-length ActRIID polynucleotide sequence such as SEQ ID NO: 4 having one or more variations, or a partially truncated version, said isolated polynucleo tide further compri sing a transcription termination codon at least six hundred nucleotides before the 3'-terminus or otherwise positioned such that translation of the poiynucleotide gives rise to an extraceliular domain optionally fused to a truncated portion of a full-length ActRiB. Nucleic acids disclosed herein may lie operably linked to a prontoter for expression, and the disclosure provides cels transformed with such recombinant polyvruceotides. Preferably the cell is a nammalian cclt such as a CHO cell In certain aspects, the disclosure provides methods for making DF Trap polypeptide Such a method may include expressing any of the nucleic acids (e g. SEQ 1D NO: 25, 27, 30 or 3 1) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cel Such a method may comprise: a) culturing a ccli under conditions stabl for expression of the GDF Trap polypeptide, wherein said cell is transformed with a GDF Trap expression construct; and b) recovringthe GDF Trap poypeptde so expressed GDF Trap 9 polypeptides may be recovered as crude, partially puried or highly puritied fractions using any of the well known techniques for obtanig protein from cell cultures. In certain aspects a GDE Trap polypeptide disclosed herein may be used in a method for promoting red blood cell production or increasing red blood cell levels in a subject, In certain embodiments, the disclosure provides methods for treating a disorder associated with low red blood cell counts or low hemoglobin levels (e g an anoemi) or to promote red blood cell production; in patients in need thereof A method may comprise administering to a subject in need thereof an effective amount ofa GDF Trap polypeptide., In certain aspects, the disclosure provides uses of GDF Trap polypeptides fbr making a medicament for the treatment of a disorder or condition as described herein, In certain aspects, the disclosure provides methods for administering a GDF rap polypeptide to a patient. In par., the disclosure demonstrates that GDF Trap polypeptides can be used to increase red blood cell and hemoglobin levels, ODF Trap polypeptides may also be used for treating or preventing other therapeutic uses such as promoting muscle growth, In certain instanceswhen administering a GDF Trap polypeptide tor promoting muscle growth, it may be desirable to monitor the effects on red blood cells during administration of the GDF Trap polypeptide, or to determine or adjust the dosing of the GDF Trap polypeptide, in order to reduce undesired effects on red blood cels. For example, increases in red blood cell levels, hemoglobin levels, or henmatocrit levels may cause increases in blood pressure. BRIEF :DESCRIPT f1N OFTHE DRAWINGS Figure 1 shows an alignment of the extracelIular domains of hurnan A ctR fA (E3Q ID NO, 15) and human ActRiiB (SEQ ID NO 2) with the residuesthai are deduced herein based on composite analysis of ntultiple ActRilB and ActRIlA crystal structures to directly contact ligand (the ligand bindrig pocket) indicated with boxes. Figure 2 shows a multiple sequence alignmnent of various verebrate ActRR !B protins and human ActRiA (SEQ ID N(Os: 16-23 Y Figure 3 shows the full amino acid sequence for the GDF Tran ActRilB(L79D 20 134)hFc (SEQ ID NO: 11), including the TPA leader sequence (double underlined), ActRIllB extracel lular doman (residues 20h134 in SEQ ID NO: I underlined and hiFc domain. The aspartate suibsihuted at position 79 in the native sequence is double underined and 10 highghted, as is the glycine revealed by sequencing to be the N-termrinal residue in the mature tfusion protein Figure 4 shows a nucleotide sequence encoding ActRIB(IL 79D5 20- 34>hbe. SEQ I) NO: 25 corresponds to the sense strand, and SEQ ID NO:3 corresponds to the antisense strand. The TPA leader (nucleodes1 -66) is double underlined, and the AcIRUiB extracellular domain (dneeotides 76-420) is underlined, Figure 5 shows the full amino acid sequence for the truncated GDF Trap ActRlB(9D 259-31)- Fc (SEQ ID NO: 26), including the TPA leader (double underlined), truncated ActRiIB extracellular domain (residues 25-131 in SEQ ID NO: 1; umderiined), and hFc domain. The aspartate substituted at position 79 in the native sequence is double underlined and highlighted, as is the glutamate revealed by sequencing to be the N terminal residue in the mature fIsion protein. Figure 6 shows a nucleotie sequence encoding ActR ilB(L79D 25- 131)-bFc SEQ D NO: 27 corresponds to the sense strand, and SEQ ID NO: 34 corresponds to the antisense strand. The TPA leader (nucleotides 1-66) is double underlined, and the truncated ActR IIB ua domain (nucleotides 76096) i underined, The amino acid sequence for the ActRLB extracellular domain (residues 25- 31 in SEQ ID NO: I) is also shown Figure 7 shows the amino acid sequence for the truncated GDF Trap Att'RB(L79D 25-31)hFe without a leader (SEQ ID NO: 28), The truncated ActRTiB extraceilulardomain (residues 25- 131 in SEQ ID NO: I) is underlined. The aspartate substituted at position 79 in the native sequence is double underined and highlighted, as is the glutamate revealed by sequencing to be the N terminal residue in the mature fusion protein, Figure 8 shows the amino acid sequence for the truncated GDF Trap ActRliB(L.79D 25-13 1) without the leader We domain, and linker (SEQ ID NO: 29) The aspartate substituted at position 79 in the native sequence is underlined and highlighted, as is the glutamate revealed by sequencing to be the N-tenninal residue in the mature fltsion protein, Figure 9 shows an alternative nucleotide sequence encoding AccR lB(L79D 25-131) hFe SEQ ID NO: 30 corresponds to the sense strand, and SEQ ID NO: 35 corresponds to the antisense strand. The TPA leader (nucleotides 1 66) is double underlined, the truncated AetRilB extracellular domain (nucleotides 76-396) is underlined, and substitutions in the wildtype nucletide sequence of the extracel luar domain are double undelined and I I highlighted (coma with SEQ ID NO: 27, Figure 6). The amino acid sequence for the AcIRliB extracdlular donain (residues 25-131 in SEQ ID NO: I) is also shown, Figure 10 shows nueleotides 76-396 (SEQ ID NO: 31) of the alterative nucleotide sequence shown in Figure 9 (SEQ ID NO: 301, The same nucleouie substitutions indicated in Figure 9 are also underlined and highlighted here. SEQ ID NO: 31 encodes only the truncated ActR IB extraceilular dornain (corresponding to residues 2513 1 in SEQ ID NO 1) with a L290 substitution eg., ActIlB(LJ9D 2-13 .) Figure I shows the effect of AtRl3(1.79D 25-13 .)-hFe on hemoglobin concentration i'n mouse mod of chemotherapy-nduced anemia Data are means ci SEM, ** P < (101 vs, paclitaxel at the same time point. This GDF Trap offset the anemia induced by paclitaxel treatment Figure 12 shows the effect of ActRiiB(L79D 25-13 1)-~hc on red blood cell (RB13C) levels in a unilaterally nephrectomized (NEPH X) mouse model of chronic kidney disease; Data are means t SEM. *** P < 11001 vs. baseline, This GDF Trap reversed the nephrectomy-induced anemia observed in control mice. Figure 13 shows the elf-ct of ActRIIB(790 25-13 }-hFc on red blood cell (R BC, hemoglobin (-lGB> and hematocrit (aT) levels W ; unilaterally nephrectom ized (NEPl-X) mouse model of chronic kidney disease Data are mean changes from baseline over 4 weeks ( SEM) * P < (105; ** P 4 0 01; *** P 4 0.001 vs. NEPIH-X controls. This GDF Trap prevented the nephrectom y-associated decline in these erythrocytic parameters, increasing each by a magnitude similar to that in kidney-intact (sham) mice, Figure 1 4 shows the efIect of ActRxiiB(79D 25-I31)-h.Fe on red blood cell (RBC) levels in a rat model of anemia induced by acute blood loss. Blood removal occurred on Day -1 with dosing on Days 0 ana Data are means + M SEN* P <001; * P < 0,001 vs' Vehicle at same time pomt, This ODE Trap improved the rate and extent of recovery from blood-loss-induced anemia. Figure 3 5 shows the effect of treatmenwith ActRll.3(L 79) 20-1 34)1hFe (gray) or ActR lB(L 9D 2131 )hFe (black) on the absolute change inlred blood cell concentration fom baseline in cynormoius monkey, VEH =: vehicle. Data are means - SEM. n 4-8 per group 12) Figure 16 shows the effect of treatment with ActRiB(L79D 20-134)-hFe (gray) or AetR I B(L790 25- 31 )hFe (black) on the absolute change in hematocrit from baseline in cynomolgus monkey, VI-- = vehicle Data are means ± SEM, n - 4-8 per group. Figure 17 shows the effect of treatment with ActRBI R9D). 20- 34 hFc (gray) or AetRI B(L79D 25-131)hFe (black) on tie abs ntie change in hemoglobin concentration forn baseline in cynomolgus monkey. VEH- vehicle Data are means ± SEN, n = 4-8 per group. Figure 18 shows the effect of treatment with ActR1lB(L79D 20-134)-hFe (gray) or AetRliB(L39D 25-131)-hFe (black) on the absolute change in circulating reticulocyte concentration forn baseline in eynomo Ius monkey. VEl-= vehicle. Data are means SEM n = 4-8 per group. DETAILED DESCRIPTION OF TH E INVENTION L Overview The transforming growth factorbeta F ( beta) superfamily contains a variety of growth factors that share common sequence elements and structural motif. These proteins are known to exert biological effects on a large variety of celi types in both vertebrates and in vertebrates, Members of the superfamil y perform iunportan fimetions during emibryonie development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesisamyogenesischondrogenesis, cardiogenesishemnatopoi esiseurogenesisand epithelial cell differentiation, The finily is divided into two general branches: the BMP/GDF and the T(F-beta/Activin/BMPl0 branches, whose members have diverse, often complementary effTets. By nanipulating the activity of a member of the TGE-beta family it is often possible to cause significant physiological ch ages in an organism, For example, the Pied montese and Belgian 31tie cattle breeds carry a loss-of-iunction mutation in the GDF8 (also called myostatin) gene that causes a marked increase in nmuse mass. Crobet et al, Nat Cenet. 1997, 17(1:7 1-4 Furthermore, in humans inactive alleges of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al N EngI J Med 2004. 350:2682-8. TGF-J signals are mediated by heteromeric complexes of type I and type II serine/threonine kinase receptors. which phosphoryrl e and activate downstream Smad 15 proteins upon ligand stimulation(Massagud;2000, Nat ReN Mol Celi Biol, 1 I 178) These type I and type receptors are transmembrane proteins, composed of a ligand -binding extraceflular domain With cysteinrich region, a transmnembrane domain, and a cyoplasmic domain with predicted serinehhreonine specificity, Type I receptors are essenital for signaling. Type 11 receptors are required for binding ligands and for expression of Type I receptors. Type I and i activin receptors form a stable complex after ligand binding, resulting in phosphorylation of Type I receptors by Type IIreceptors. Two related Type I receptors (ActRII), AdcRIIA and ActRIIB, have been identified as the Type receptors for activist (Mathews and Vale. 1991Cel 65:973-982; Attisano et at. 1992, Cell 68: 97-108), Besides activist ActRIUA and AciR 11B can biochemicaly interact wit several other TGFI family proteins, including BMP7, Nodal, GDF8, and GuDI I (Yamashita et al - 995, 3 Cell Bio, 130;27-226; Lee and McPherron, 2001 Proc. Nath Acad. Sci 98930693 1i Yeo and Whitman. 2001, Mol. Cell 7; 949-957; Oh e aL, 2002, Genes Dev. 16274954) ALK4 is the primary type I receptor for activins, particularly for activin A, and AL K-7 may serve as a receptor for activins as well, particularly for activin B, in certain embodiments, the present invention relates to antagonizing a ligand of ActR I1B receprs alsoo referred to as an ActR 111B igand) with a subject GDF Trap polypeptide Exemplary ligands of ActRU B receptors include some TGF 0 family members. such as activin, Nodal, GDF8, GDF I Land BMPR Activins are dimeric polypeptide growth factors that belong to zhe TOF-beta superfarnily There are three prncipal activin f6rms (A, B, and AR that are homo/heterodimers of two closely related P subunits (PAP P3, and PA respectively). The human genome also encodes an acuivin C and an activin Evhich are primarily expressed in the liver, and heterodimeric forns containing p or p are also known, I the TOE-beta superatnily, activins are unique and multifunctional feeors that can stimulate hormone production in ovarian and paCena cells, support neuronal cell survivalnfluence cel-cycle progress positively or negatively depending on cell type and induce mesoderrra differentiation at least in amphibian embryos (DoPaolo el aL, 1991 Proc Soc Ep Biol Med 1985004512- Dyson et al., 1997, Curr Bio. 81-84; Woodruff, 1998. Biochem Pharmacol. 55:953963) Moreover, erythroid difterentiation factor (EDF) isoated &om the stimulated human monocytic leukemic cells was found to be identical to activin A (urata et aL, 1988. PNAS, 85:2434) It has been suggested that activin A pronotes ertyhopoiesi in he bone 14 marrow In several tissues, activin sgnal ing is antagonized by its related heterodirmer inhibin. For example, during the release of licle-stimulating hornone (FSH) from the pituitary, activin promotes FS1H secretion and synthesis, while inhibin prevents FSH secrettion and synthesis. Other protein that nay regulate activin bioactivity and/or bind to activin include foilistatin (FS) folistati related protein (FSRP) and a macrogbuli. Nodal proteins have functions in mesoderm and endoderm induction and formation, as well as subsequent organization of axial structures such as heart and stomach in early embryogenesis. It has been demonstrated that dorsal tissue in a developing vertebrate embryo contribute predominantly to the axial structures of the notochord and pre-chordlal plate while it recruits surrounding cells to form non-axial tembryonic strctures Nodal appears to signal through both type I and type H receptors and intracellular effectors known as Smad proteins Recent studies support the idea that ActRiA and ActRlB serve as type I receptors for Nodal (Sakuma et ai,, Genes Cells. 2002 7:401-12). It is suggested that Nodal ligands interact with their co-factors (e.gcripto) to actiate activin type I and type 11 receptors, which phosphorylate Smad2, Nodal proteins are implicated in many events critical to the oarly vertebrate embryo, including mesoderm formation anterior patterning, and left right axis specification. Experimental evidence has demonstrated that Nodal signaling actiates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TOF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to hone morphogenetic proteins. Recent i-csuIts provide direct biochemical evidence that Nodal signalng is mediated by both activin-TGF--beta pathway Smrads. Smad2 and Smada. Further evidence has shown that the extracelldar cripto protein is required for Nodal signaling, making it distinct from activin or 'GF-beta signaling. Growth and Differentiation Factor-8 (ODFS) is also known as myostadn. GDF8 is a negative regulator of skeletal uscle mass. GDP8 is highly expressed in the developing and adult skeletal muscle. The GDF8 null nation in transgenic mice is characterized by a marked hypertrophy and hyperplasia ol the skeletal muscle (McPherron et al., Nature, 1997, 387.1390 Similar increases in skeletal muscle mass are evident in naturally occurring mutations oftGD)8 i catte (Ashmore et all.1?4. Growth, 38501-507; Swatland and Kieffer J. Anm Sci 1994, 3852757 McPherron and Lee, Proc. Nal Acad. Sci. USA, 1997, 94:1245712461and Kambadur et at Oenome Res 1999. 7010915) and, strikingly, in humans (Schuelke et a, N Engl J Med 2004;350:26828) Studies have also shown that 15 muscle wastng associated with IIinfectiorn liumans isaccompanied by increases in GDF8 protein expression(GonzalezCadavid et al PNAS, 1998 95:14938443) In addition, GDF8 can modulate the production of nmscle-specific enzymes (eg,, creatinekinase) and modulate myoblast cell proliferation (W() 00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8 domain dMier iis biological activity (Miyazono et al (988) bBiol Chem, 263; 6407-6415; Wakeield e al (1 988) J. Bio Chem,263; 76467654; and Brown et a (Il 990) Growth factors, 3: 3543)e Other proteins which bind to GDPS or structurally related proteins and inhibit their biological activity include follistatin and potentially, iistatinseated proteins (Gamer t al (1999) Dev. BioL, 208: 222-232) Growth and Differentiation Factor--I (CDP 1 V. also known as BM P 11 is a secreted protein (McPherron et aL, 1999, Nat Genet. 22: 26 -264). GDFI I is expressed in the tail bud, linb bud, maxillary and mandibular arches and dorsal root gariglia during nouse development (Nakashina et at, 1999. Mch. Dev 80:185-189), GDP 1I plays a unique role in patterning both rmesodennal and neural tissues (Gamer et al 1999, Dev Biol, 208:222 32) G IDF I was shown to be a negative regulator of chondrogenesis and nyogenesis in developing chick limb (Garner et at 2001. Dev Biol. 229:40720), The expression of GDT I in muscle also suggests its role in regulating muscle growth in a similar way to GDFS In addition, the expression ofCDFI I in brain suggests that GDFJ I may also possess activities that relate to the function of the nervous system iteresti ngtGDP, 1 was found to inhibit neurogenesis in the olfactory epithelium (Wu et at 2003, Neuron. 37T1972207). Hence, GDFI I may have in vro and in vivo applications in the treatment of diseases such as muscle diseases and neurodegenerative diseases (e g amyotrophic lateral sclerosis). Bone niorphogenetic protein (BM P7), also called osteogenic protein- (OP--) is well known to induce cartilage and bone fomiation. In addition, BMP7 regulatesa wide aray of physiological processes for example. BM P7 may be the osteoemductive tctor response for the phnmeo of epiv~thelial osteogenesis. It is also found that BMP7 plays a role in calcium regulation and bone honeostasis. Like activin, MP7 binds to Type If receptors, ActRIIA and AcIR1IB-lowever BMP7 and activin recent distictype I receptors into )heteronmerie receptor complexes. The major BMP7 ype I receptor observed was ALK2, while activin bound exclusively to ALK4 (ActRilB). BM-T and acivin cI icited distinct 16 biological responses ard activated different Smad pathways (Macias-Silva et al. 1998, J Biol Chemn 273 256285*6) As demonstrated herein, a GDF Trap polypeptide, which is a variant ActRiB polypeptide (AcRIIB), is more effective at increasing red blood cell levels in nivo as compared to a wild-type soluble ActRifB polypeptide and has beneficial effects in a variety of models foranemias. It should be noted that hematopoiesis is a complex process, regulated by a variety of factors, including erythropoietin, -CSF and iron homseostasis, The terms "increase red blood cell leves" and "promote red blood cell frmation" refer to clinically observable metric such as hematocrit; red blood cell counts and henoglobin measurements, and are intended to be neutral as to the mechanism by which such changes occur, in addition to stimulating red blood cell levels, GDP Trap polypeptides are useful for a variety of therapeutic apications, including fOr example, promoting muscle growth (see PCT Publication Nos. WO 20061012627 and WE 2008/097541, which are hereby incorporated by reference in their entirety), in certain instanceswhen administering a GDP Trap polypeptide for the purpose of increasing muscle, it may be desirable to reduce or minimize effects on red blood cells. By monitoring various hematologic parameters in patients being treated with, or who are candidates for treatment with, a GDF Trap polypeptide, appropriate dosing (including amounts and frequency of administration) may be determined based on an individual patients needs baseline hematologic parameters, and purpose for treatment. Furthermore, therapeutic progress and effects on one or more helmatologic parameters over time may be useful in managing patients being dosed with a GDF Trap polypeptide by faciltating patient care, determning appropriate rain tenance dosing (both amounts and fr'equen cy). etc. The terms used in this specification general have their ordinary meanings in the art, within the context of this invention and in the speci ontext where eacb term is used. Certain terns are discussed below or elsewhere in the specification, to provide addit ional guidance to the practitioner in describing the compositions and methods ofthe invention and how to make and use them: The scope or meaning of any use ofta term wjll beapparent from the specific context in which the term is used. "About" and "approximately" shall generally nean an acceptable degree of error for the quantity measured given the nature or preci sion of the measurments. Typi calmly 17 exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of vales, Altemativel y, and particulay in biological systemsthe terms "about" and approximatelyl" may mean values that are within an order of magntude, preferably within 5 fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate Unless stated otherwise neaning that the term "about" or "approxiiatel y" can be infrred when not expressly stated. The methods of the invention may include steps ofcomparing sequences to each otherncluding wild-type sequence to one or more mutants (sequence variants). Such comparisons typicall y comprise alignments of polymer sequences e g, using sequence alignment programs and/or algoridthms that are well known in the art (for exanpe, B LAS. FASTA and MEGALGN to name a few) The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce "gap" (typically represented by a dash, or "A") in the polymer sequence not containing the inserted or deleted residue, "H-lomologous, in all its grammatical forms and spelling varationss, refers to the relationship between two proteins that possess a "common evolutionary origin." including proteins forn superfamilies in the same species of organism as well as homologous proteins frnm different speces of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected their sequence similarity; whether in terms of percent identity or by the presence of specific residues or montfs and conserved positions. The term "sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a comm on evolutionary origin. However, in connon usage and in the instant application, the term 'homologous when modifed with an adverb such as "highly." may refer to sequence similarity and may or may not relate to a common evolutionary origin, 2 DF Trap Polypeptides In certain aspects, the invention relates to GDF Trap polypeptides. e soluble vacant ActR 1 B polypeptide including, for example, fragment, functional variants, md Is modied f orms of AcTRUB polypeptides. In certain embodiients the GDF Trap ptides have at least one similar or same biological activity as a corresponding wild type ActRJIB polypeptide. For exam ple, a GDF Trap polypeptide of the invention may bind to and inhibit the function of an ActRIIB ligand (e g activin A vin AB, activin B, Noda, GDFS, GDF1I or BIMP7). Optionally a CDF Trap polypeptide increases red blood cell levels. Examples of GDF Trap polypeptides include human Ac-tRIB precursor polypepti des (SEQ ID NO: I or 39) having one or more sequence variations, and soluble human ActRllt polypeptides (e.g, S EQ ID NOs 2, 3, 7, 11, 26, 28, 29, 32,37 38 40 and 41) having one or more sequence variations. A GDF Trap refers to an Actki13 polpeptide having a decreased affinity for activin relative to other ActRIB ligands. including for example GDP II and/or myostatin. As used herein, the term 'ActR IUB refers to a fainily of activir receptor type 1b (ActR1lB) proteins frorn any species and variants derived from such ActIRIB proteins by muitacenesis or other modification. Reference to ActR IIB herein is understood to be a reference to any one of the currently identified forms. Members of the ActRUB family are genera ly transiembrane proteins composed of a igandbinding extracellular domain with a steine-rich region tranmembrane domain and a cytoplasmc doMnain with predicted serine/tireonine kinase activity Amino acid sequences of hum an AetRUIA sohule extracelular domain (provided for comparison) and ActRIHB soluble extracellhlar domain are illustrated in Figure I The term "'ActRHB potypeptide' includes polypeptides comprising any naturally occurring polypeptide of an ActRilB family member-as welnl as ny variants thereof (including mutants fragments, fusions, and peptidomimetic forms) that retain a useful activity. See, for example. W( 2006/Ql26i27 For example, ActRIlB polypeptides inchade? polypeptides derived from the seence of any known ActR 11B having aleast about 80% dentica to the sequence of an ActR IIB polypepfide, and optionaiy at least 85%, 90% 95% 97%. 99% or greater identity For example, an ActR11B poiypeptide may bind to and inhibit the function of an ActR 11B protein and/or activi. An ActiklB polypeptde which is a GOF Trap may be seleted for activity in promoting red blood cell forniaticn in Inv.o Examples of AciRiB polypeptides includehman ActRIlB precursor polypeptide (SEQ ID NO: I and 39) and soluble human ActR 118 polypeptides (e.g, SEQ ID NO: 2, 3. 7. 1, 26, 28, 29,32,37,38, 40 and 41 ) Numberng of amino aids for all ActRllB-related 19 polypptides described herein is based on the mnbering ibr SEQ 1D NO , utdes S pWcIcahIIy (letsiegn aotd othewrse, Itoe human ActRHI3 precursor pnxemr sequence is asflos NTAPWVALALLWC$L P05 GRGEAETRECIYYWNANWELER4 , ,QSGLERC ZGEQDKPRCYAStWP{S SGTIELVXKGC-WLDDFNCYDRQECVATEENPQ VY FCCCEGNFCNERFTHLPEAGGPEVTYEPP PTAPT LLTV LAY S iF IG LISLVLLAWMYPPK>2GHVDIMEPGPPEPSPLVLKPLQLET.7 A KGRFGCVWV.AQLMN DFVAVK IPPLQ DKQSWQS}LRE 'USMKHNLL Q IArGNEEWLTFDGLDLAGNIITWNLCUVA&T CRGL 145 V CFEG PS IANKDPKSSN VLLKS DLIIAVLAFG.A \Jfr rl PGKP(:rTHc;QVtir;RYMA PSVLEGAIN FQRLATL SI MYAMGLV . 1.WSLVSRCLRADGPVDEYMI. EEIG V EELEVVHtKMRG'P71K DHWLRR PGLAQLC VTIEECW PH AEPLzGCEEVSLURR$VuTTS DCLSLATSTNVLPPSESI(SE---Q 11D) NO:- I) The sigalP peptide, is single urtderlnet the extracellular doinair. is in-bold and the potentiW Niinked gyoytinsites are in bxs A ormiiwith an alanine at no.-siton 64 is also reported in the literatureas follows: MTA PWVALA 1LG SLW PG 5GRGEAZTT4ZC1YYNAWELER~QSG.LERC EGEQKRIICYAW4jSSGTIELVKKG!CWILDDFltCYDRQECVATEENPQ V'YFCCCEGNFCNERFTHLPF.AGGPEVTYEPPPTAPTLLTV LAYCL L P 10' GL1 3 IL AF YLRP YHVDIitEDPcOPPPPSPLVCLP:PLQLLEIK ARCYRP(CVWKAQLMANDFVAVKI rsP nV~a% 4E~Inz~r'cCMrA ENT X QSIKABKRGSNLEVELWLITA F455cS LOrKGN1 ITWNLLCHVAETN VRVBFPCK P P{THCQVGTRRYRIAPEVLEGAINFQRDAFLRI CRYARC LV LW'ELVSRCKAACO PV'D""YMLPV EEGQ;t5ZELO&YwKKRPTIK DHWLKH PCLAQLC VT IIEC. EGNOSAR LCAGCVVES VSL I S.S V}GTT S CCLVSLVTSVTNVDBLPPKE.SS.1 (SfE 10 NO: 39) 20) The human ActBR 11 solub I (extrace I ular), processed poI ypept ide sequence is as follows: eGRGEALTPNECIYYNAN WEL ERTNQSGLERCEGQDERINCASWRNSSG T LONLTTHEP E~G~gF~g(SEQ ID NON). 2 The alternative ftrm with an A64 is as allows: GRGLAETRECi YNANWLElkRTNQSGIEPCEGEQUKRLHC AStSANSSG T I ENGGWE DNCYDEQELVEENPQVYFCCCE NERFT HI? EAggGGPLVYPI+IvP (SEQ ID NO: 40) In some condition, the protein may he produced with an St sequence at the N tein us TI he (-term in al "tad o the extraceth Fuar domain is u order Ined, The sequence with the "tail" deleted (a A15 sequence) is as follows: GRGEAETREC YYNANW ELERTNQSGL ERCEGEQDKRLH CYASWRN S(G TI ELVKKCWL DENCY DRQEVAT E EEQVY £90EG L FCER f F. VL T EA (SEQ ID NO: 3) The alternative form with an A64 is as flows: GRGEAETPR CIKYNANWELEPRTNQSGLLERCEGLQDKPIHCYASWANSSG T IELNK KGWL DFNCY DRO VATEEN PQV YF CCEG NLF N E P FTHLP EA (SEQ ID NO: 41) In some conditions, the protein may be produced with an "SUE sequence at the N teminus, The mucleic acid sequence encoding a human ActR IIB precursor protein is as follows: (nuleotides 5 1543 of Genbank entry NM 001 106)(the sequence as shown provides an alanine at position 64, and nay he modified to provide an arginine instead) A TGACGGCGCCQCTGGGTCTCGCCCTCCT CTGGGG ATC GOTGTGGCG OCCGTGC7GC GGGCCCnAGGC TCAAGAACG AQTGA TCTACT ACAA GAAGGGAGCAGAOAACGGT cC .GOCGCTCTGCCAAGA CAACTGCTAGGATAGGCAGATTTGCGGAACC~A CN tN G4A' GT~CTTCTG CGTGTGACAAOMTTCGOACACGCTTCATC AT T TIGCA G A G-,:jC TG GGGG GG AAGiST CA G T A CA ISCCACCA AT?21 AGCCCCCACCCCTCACGGTG ;TGCCTACTCACTGCTGCCCATGGG A TCGCCTGTGGCT TTTGG33.-ATGTACCGGC TGC AGCCCCCCTACG4GTCATGT GGACATCWeCATGAGGACCQT GGGCCTCCACY: ACCTCCCTTGGGCTGAAiGCAGAGGCfiTGG&A3GA TCAA GGGGGT -T CGGT G GC GGGCCA3TATAAGc 'TTGTAGCTGTCAAGATCT"TC CACT CCAGGACAAGCAGCTGGCAGAG A -P": I TGACGGTCATCCC ArCACETGCAT AT AGCACAGAACCTCA GG"GKT CGT3 CGCe CGGGAACATCATCACAT CCAACGAACTG 3T~~st ACAACAAAGATG TCACCAGGCCTCTCAT ACCTGCATGAGGCATGTCCCCTGGTGCCGTGGCG C. S SEQ D N(,- 4 AGGCCaACACCG TTATTGCCACfGACTTTAAAAGTAA GAATG Tkpd ATTCGA'J TAGCCCTC-t-ACAG CTCTCTGA ACC(GTGCT GCACGAGACGGTACATGGCCGAGGTGC.GAGGAGCCACAACT GCAGAGAGA-.TGCCTTCCTG CGCATTGAC:ATGTATGCCATGGG~trTGT CTLijTGGKGAK.~GCTTGGTCUGCGCAGGCGCAACGAC CGTGGATGC AG TACATGC CCT GAGGAAGAGATT GCCAGCACCCTTCG T GGA GGAGCTGCAGAGTGGCTGGTCACA AGAAGATGAGGCCCACCATTAAA CATCAxyCGTTGAAACv'-ACCCGGCTGGECC KAGCTT. .GTGTGACG'ATCG AGGAGTC'GCGACGAATGCAGAGGCTCGCTTGTkCGCGGGCTG{3KKiOK(1-TT GGAGGAGCGTGTGCCCTGATTCGAGGTCGCTCAACGCA "ACGC

K-C

7 TAGAGT{AAGK KA~ tCTA (sEQ IDNO: 4) .- h nuCleic aeid sequendenCncoChdnza humn ACtkBA soubble (extracehiar) polypepude is as follows (the sequence as shown provides an alaie at position 64. and may be modified to proid aarginine instead: C7CCA CTGC CCCC CCAA AGCGG . C AGCGCTGGG>N GCGA GCAG GACAAC GC TGC ACT GC TA COP C CT GGCC AAC AGO T7 TC ACCATOGAC T PC AAG AAG''GCTGOCOC OP GA'' TGA T~ 7CA ATG2 ~ 'zzr4; ~ \C'~CC .- 2' GAGGCT CGGGGC CCAAGTCACGTA CGAGCCACCCCCGACAGCCCCZA CC (SEQ ID NO: 5) In a specific embodiment, the invention relates to ODF Trap polypeptides which are variant forns of sol uble AetR 1113 polypeptides As described herein, the term soluhle ActRilB poiypeptide generally refers to polypeptides compsing an extracellular domain of an ActRilB protein. The term sluble AcIR UB polypeptideas used herein includes any naturally occurring extracellular domain of an ActRi protan as well as any variants thereof (including mutants, fragrents and peptidomimetic forms) thatretain a useful activity For example, the extracellular domain of an ActRIlB protein binds to a Iigand and is eneraly soluble. Examples of soluble AcIRIBs polypeptides include ActRIJEB soluble polypeptides (eg, SEQ ID NOs: 22 3 7, 11, 26, 28, 29. 32,37 38 40 and 4 1), Other examples of soluble ActRUB polypeptides compise a signal sequence in addition to the extracellular domain fan ActRUB protein, see Example I, The signal sequence can be a native signal sequence of an ActRUIB, or a signal sequence fiorn another protein, such as a tissue phsminogen activator (T PA) signal sequence or a honey bee melitin (11DM) signal sequence. The disclosure identifies functionally active portions and variants of ActRIlB, Appicants have ascertained that an Fe fusion protein having the sequence disclosed by Hilden et al, (Blood. 1994 Apr 1 5;83(8):21 670), which has an Alanne at the position corresponding t ao am acid 64 of SEQ ID NO: I (A64) has a relatively low affinity for activin and GF11 By contrast, the same Fc fusion protein with an Arginine at position 64 (R64) has an affinity fr activin and GDF-li in the low nanomolar to high picoiolar range Therefore a sequence with an R64 is used as the wildtype reference sequence ,or human ActRitB in this disclosure. Atisano et a (Cell 1992 Jan 10E )697 10) showed that a deletion of the prolirne knot at the (-ternnus of the extracellular domain of AcRtiB reduced the affinity of the receptor for activin. An ActRilIFe fusion protein containing aino acids 20119 of SEQ lD NO 1, AtR I IB(20 i 19)~Pc has reduced binding to GD1% I and activin relative to an ActRilB(20-134>-Fe, Mich includes the proline knot region and the complete juxtamenibrane domain. However. an ActRiB(2029)-Fc protein retains similar but 2 J, somewhat reduced activity relative to the wild type, even though the proline knot region is disrupted. Thus, ActRl1B extracellular doinains that stop at arirno acid 134 1 33 13 2, 131, 130 and 129 are all expected to be active, but constructs stopping at 134 or 133may be most active, Similarly, mutations at any of residues I 29- 1 34 are not expected to alter i gand binding affinyby large margins. In support of tsrutafions of P129 and P130 do not substantially decrease ligand binding. Therefore, a (3D FTrap polypeptide which is an ActRlIl3Fc fusion protein nma end as early as amino acid 109 (the final cysteine) however, forms ending at or between 109 and 119 are expected to have reduced ligand binding, Amino acid if) is poorly conserved and so is readily altered or trncated. Forms ending at 1 28 or later retainligand binding actiity Forms ending at or between 119 and 127 will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental setting. At the N-ermiraus of ActRiB, -it is expected that a protein beginning at amino acid 29 or before will retain ligand binding activity. Amino acid 29 repress the iriitial cysteine, An alanine to asparagine noutation at posdiotin 24 introduces an N t -inked glycosylation sequence t substantially affecting ligand binding, This confirms that mutations in the region between the signal Cleavage peptide and the cysteine cross-linked region, corresponding to amino acids 20-29 are well tolerated, In particular, constructs beginning at position 20, 21 22, 23 and 24 xviii retain activity, and constructs beginning at positions 25, 26. 27, 28 and 29 are also expected to retain activity. Data shown in the Examples demonstrates that, surprisingly, a construt beginning at 22, 23, 24 or 25 will have the most activity. Taken together, an active portion of ActRUIB comprises amino acids 29-109 of SEQ I D NO; I and GDP Trap constructs may, fo example comprise a portion of ActR B beginning at a residue corresponding to anino acids 20-29 of SEQ ID NO. I or 39 and ending at a position corresponding to amino acids 109 134 of SEQ ID NO; I or 39. Other exainpies include constructs that begin at a position from 20-29 or 2129 and end at a position from 119-134, 119-113,.129-I34 or 129-I33 of SEQ ID NO; I or 39. Other examples ndude constructs that begin .it a position firomn 20-24 (or 21-24, or 22-25) and end at a position from 109- 134 (or 109-133), 119-134 (or 119-133) or 129134 (or 129-133) of S EQ iD NO; 1 or 39 Variants within these ranges are also contemplated, particularly those having at least 80%, 85%, 90%. 95% or 99% identity to the corresponding portion of SEQ ID 24 NO: I or 39 In certain embodiments, the GDF Trap polypeptide comprises, consists essentially of or consists of a polypeptide having an amino acid sequence that is at least SO0N % 9 95% 96% 97% 99% or l00% identecal to amino acid residues 25 13 1 of SEQ ID NO I or 39. In certain embodinents, the GD? Trap pcdypeptide comprises, consists essentially of or consists of. a polypeptide having an amino acid sequence that is at least 80%, 85%, 90%. 95% 96%, 97%, 98%, 99% or 1 00% identical to SEQ I) NOs: 7, 26, 28, 29, 32, 37 or 38, In preferred embodiments, the GDF Trap polypeptide consists of, or consists essentially of the amino acid sequence of SEQ I) NO: 7, 26, 28, 29, 32. 37 or 38 The disclosure includes the results of an analysis of composite ActRIllB structures, shown in Figure 1, demonstration that the ligand binding pocket is defined by residues Y31, N33, 135, L38 through T4 1, E47 E 0 Q53 through K55, 1-57, H-58I Y60, S62, K74, W78 through 8, Y85, R871 A92, and E94 through F 101 At these positions, it is expected that conservative mutations will be toleratedadthough a K74A mutation is welltoierated, as are 140A, K55A, F82A and mutations at position L79. R40 is a K in Xenopus, indicating that basic amino acids at this position will be tolerated. Q53 is R in bovine ActRIIB3 and K in Xenopus ActRIIB, and therefore arniio acids including R, K Q N and H will be tolerated at this position, Thus a general formula for a GD? Trap protein is one that conrises amino acids 294 09 of SEQD NO: 1 or 39, but optionally beginning at a position ranging fom 20~ 24 or 2225 and ending at apositon ranging fron 129434, and comprising no more than I 2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and zero, one or more noneonservative aherations at positions 40, 53, 55, 74, 79 andQor 82 in the ligand binding pocket, Such a protein may retain greater than 80%, 90%, 95% or 99% sequence identity to the sequence of amino acids 29109 of SEQ ID NO: I or 39 Sites outside the binding pocket, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain (as noted above), and positions 4246 and 6573, An asparagine to alanine alteration at portion 65 (N65A) actually improves hgand binding in the A64 background, and is thus expected to have no deimenal effect on Igand binding in the R64 background. This change probably eliminates glycosylation at N65 in the A64 background, thus demonstrating that a significant change in this region is likely to be tolerated. While an R64A change is poorly tolerated, R64K is welktolerated, and thus another basic residue, such as H may be tolerated at position 64. 25 AcR1B is wel-onserved across nearly all vertebrates, with large stretches of the extracellhar domain conserved completely; Many ofthe ligands that bind to AciUlB are also hi ghy conserved. A coordin g) y, comparisons of ActR iIB seq fences from vari ous vertebrate organisins provide insights into residues that may be altered, Therefore, aractive, hunan AetRU1B variant polypeptide useful as a ODF Trap may include one or more amino acids at corresponding positions Fromi the sequence of another vertebrate ActRI18 or may include a residue that is siunlar to that in the human or othervertebrate sequence The flowing examples ilhstrate this approach to defining an active ActRUB variant 4 is a valine in Xenopus ActRIHB, and so this position may be altered, and optionally may be altered to another hydrophobic residue such as V. 1 or F, or a non-polar residue such as A. E52 is a K. in Xenopus that's site may be tolerant of a wide variety of changes, including polar residues, such as ED KQ Q R, t ST, P, Y and probably A. T93 is a K in Xenopus indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, K It, 1) H, (3 t G and Y, l08 is a Y in Xenopus. and therefore Y or other hydrophobic group, such as , or L. should be tolerated. El I is K in Xenopus, indicating that charged residues will be tolerated at this posifio. including D, R K and H, as well as Q and N, RI 112 is K in Xenopus, indicating that basic residues are tolerated at this position induding R and 1-L A at position 1! 9 is relatively poorly conserved, and appears as P in rodents and V in Xenopus, thus essentially any annno acid should be tolerated at this position. The disclosure demonstrates that the addition of a further N-linked glycosylation site (N-N-SAD increases the serum half-ife of an ActRilUBFc fusion protein, relative to the ActRilB(R64)Fc for. B0y introducing an p at position 24 (A24N construct), an NXT sequence is created that confers a longer half-life, Other NX(T/S) sequences are found at 42-44 (NQS) and 65-67 (NS), although the latter may not be efficiently glycosylated with the R at position 64. N-N-Sfi' sequences may be generally introduced at positions outside the ligand binding pocket defined in Figure L. Particularly suitable sites for the introduction of non-endogenous N-N-SIT sequences include amino acids 2029, 20-24, 22-25, 109-134, 120 134 or 129134 N-X-S sequences may also be introduced into the linker between the ActR II B sequence and the I- or other fusion component. Such a site may he introduced with miininal effort by introducing an N in the correct position with respect to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N, Thus, desirable alterations that would create an N-linked glycosylation site are: A24N R64N,S60N 26 (possibly combined with an N65A alteraion),El 06N IR 112N, l200N, E123N P129N, A132N. R I12$ and R112 LAny S that is predicted to be glycosylated may be altered to a T without creating an imunoer ic site, because of the protection afforded by the gycosylation. Likewise, any T that is predided to be glycosylated may be alered to an S. Thus he alterations S67T and S44T are contemplated Likewise in an A24N variant an S26T alteration may be used, Accordingly a iGDF Trap may be an ActR 111 variant having one or more additional nonwendogenous Nlinked glycosylation consensus sequences. Position L79 of ActRilB may be altered to confer altered activin -- myostatin (GDF 11) binding properties. L79A or 139P reduces D&I I binding to a greater extent than actvn binding. L79E or L79D retains ODEl I binding, Remarkably the L79E and La9D variants have greatly reduced activin binding. hM vivo experiments indicate that these non activin receptors retain significant ability to increase red blood cells but show decreased effects on other tissues. These data demonstrate the desirability and feasibility for obtaining poiypeptides with reduced effects on activin. In exemplary embodimients, the methods described herein utilize a GDF Trap polypeptide which is a variant ActRUB polypeptide comprising an acidic amino acid (eg, D or E) at the positin corresponding to position 79 of SEQ ID NO: I or 39, optionally in combination with one or more additional anino acid substitutions. additions. or deletions. The variations described may be combined in various ways. Additionally the results of the mutagenesis program described herein indicate that there are amino acid positions in ActRIIE that are often beneficial to conserve These include position 64 (basic amino acid), position 80 (acidic or hydrophobic ammo acid), position 78 (hydrophobic and particularly tryptophan), position 37 (acidic, and particularly aspanic or glutamic acid). position 6 (basic anino acid), position 60 (hydrophobic anmino acid, particularly phenylalanine or tyrosine), Thus, in each of the variants disclosed herein, the disclosure provides a framework of aino acids that may be conserved. Other positions that may be desirable to conserve are as follows: position .52 (acidic amino acid) position 55 (basic amino acid) position 81 acidice), 98 (polar or charged, particularly E D, R or K), In certain embodiments, isolated fragments of ActRI1B polypeptides can be obtained by screening polypeptides recominantly produce ed from the corresponding frangment of the nuceic acid encoding an ActRIMB poypepid (en.g. SEQ ID NOs: 4 and 5) In addition, fragments can be chemical synthesized using techniques known in thc art such as 27 conventional Merrifield solid phase fMoc or t-Boc chemistry, The fragments can be produced {recombinantly or by chemical hesis) and tested to identify those peptidyl agentss that can function, for example, as antagonists (inhibitors) or egoists (activators) of an ActRIIB protein or an ActR1IB ligand. In certan embodirents, GDF Trap polypeptide is avariant AcIRliB polypeptide having an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 2,3,7, 11, 26, 2S 29) 32,37. 3,8 40 or 41. I certain cases, the GDFyap has an amino acid sequence at least 80%, 85% 90%. 95% 97% 98% 99% or 100% identcal Io an amino acid sequence selected from SEQ ID NOs: 2, 3 7 11 , 2 82 29, 32,7 38, 40 or 41 in certain emobdiments, the GDF mrap comprises, consists essentially of or consists of an amno acid sequence at least 80%. 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ 1D NOs: 2. 3, 7, 11, 26. 28, 29, 32, 38, 40 or 4 1 wherein the position corresponding to L79 of SEQ ID NO: I is an acidic amino acid (eg a D or E amino acid residue), In certain ernbodinents, the present invention contemplates making functional variants by modifying the structure of a GDF Trap polypeptide for such purposes as enhancing therapeutic efficacy, or stability (eg ex vivo shelf life and resistance to proteolytic degradation in viv GDEf Trap polypeptides can also be produced by amino acid substitution deletion, or addition For instance t is reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threoninewith a scnne. 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 with a famBiy of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a GDE Trap polypeptide results in a functional variant can be readily determined by assessing the ability of the GDF 'rap polypeptide to produce a response in cells relative to the unmodified GDF TRap polypepride or a wild-typeActRiB polypeptide, or to bind to one or more igands. such as activin, GDF I I or myostatin as compared to the unmodified GDFrap polypeptide or a wild-type ActRH B polypeptide, in certain specific embodiments, the present invention contemplates making mutations in the extracellular domain (also referred to as ligand-binding domain) of an ActR MB poiypeptide such that the ActRlB polypeptide has altered igana finding activities 28 (esg, binding affiity or binding specifieity) in certain casessuch GDF Trap polypeptides have altered (elevated or reduced) binding affinity for a specific ligand In other cases, the GDF hrap polypepides have altered binding specificity for A ctR UB ligands. For example, the disclosure provides ODF Trap polypeptides that prcferentialiy bind to GDF8/OD F I relative to activins, The disclosure further establishes the desirability of such polypeptides for reducing off-target effects, although such selective variants may be less desirable for the treatment of severe diseases where very large gains in red blood cell levels may be needed for therapeutic effect and where some level of offtarge effect is acceptable For example, amino acid residues of the ActR 1B protein, such as E39, K 5 O,60 K04, W78, D805 and F101, are in the ligand-binding pocket and mediate binding to its ligands such as activinand GD8 Thus, the present invention provides a GDF Trap comprising an atred ligandbindinig domain (e.g, GDF-binding domain) of an ActRIlD receptor, which comprises one or more mutations at those aminio acid residues Optionally, the altered ligand-binding domain can have increased selectivity for a ligand such as GDF8 relative to a wild-type ligand-binding domain of an ActRilD receptor. To illustrate, these mutations increase the selectivity of the altered Iigand-bindirg domain for ODFS over activin. Optionaily, the altered ligand-binding domain has a ratio of 1K for activin binding to d for GDF8S binding that is at least 2,5. 10, orneven 100 fold greater relative to theratio for the wild-type liigand-binding domain. Optionally, the altered ligand-binding domain has a ratio of ICs for inhibiting activin to Q, for inhibiting ODF8 that is at least 25, 10, or even 100 fold geater relative to the wild-type lgand-binding domain. Optionally, the altered ligtand binding domain inhibits GDFS with an IC: at least 2, 5, 10, or even 100 times less than the lCsa for inhibitirng activin. As a specific example, the positively-charged amino acid residue Asp (DSO) of the ligand-binding domain of ActR11 B can be mutated to a diferen amino acid residue to produce a GDF Trap polypeptide that preferentially binds to GDF8, but not actvin. Preferably, the DS residue is changed to an amino acid residue selected from the group consisting of: an uncharged amino acid residue, a negative amino acid residueand a hydrophobic amino acid residue. As a further specific example, the hydrophobic residue, L)9, can be altered to the acidic amino acids aspartic acid or g utamic acid to greatly reduce ativin binding while retaining GDF 1 binding As will be recognized by one of skill in the art, most ofthe described mutations, variants or modifications may be made at the nucleic 29 acid level or in some cases, by post translational modification or chermiea synthesis, Such techniques are well known in the art. In certain embodiments, the present invention contemnplates GDF Trap polypeptides having specific mutations in ActRllB so as to alter the glycosylation of the Act RI[B polypeptide. Exemplary glycosylation sites in (hF Trap polypeptides are illustrated in Figure I (egthe undefined NX(ptT) sites) Such nmutations may be selected so as to introduce or elimmane one or more glycosyltion sees, such as Olinked or N-hnked glycos tes, Asparagine-linked glycosylation recognition sites generalIy cormpie a tripeptide sequence, asparagi ahreonine (where "X" is any amino acid) wv which is specifically recognized by appropate cellular glycosylation enzymes. The ateration nay also be made by the addition of or substitution by one or more serine or threonine residues to the sequence of the wild-type AcRIlIB polypeptide (for Oinked glycosylation sies). A. variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or ammo acid deletion at the second position results in non-glvcositation at the modified tripeptide sequence Another means of increasing the number of carbohydrate moieties on a GDF Trap polypeptide is by chemical or enzymatic coupling of gyeosdes to the GDF Trap polypeptide, Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulhydryl groups such as those of cysteie;(d) free hydroxyl groups such as those of serine, threonine, or bydroxyproline; (e) aromatic residues such as those of phenylaanine, tyrosine, or tryptopn;or (f) the anmide group of glutamine lhese methods are described in WO 87/05330 and in Aplin and stond1981) CRC Crit Rev. Biochem., moieties present on a ODF Trap poiypeptide may be acconplished chemically and/or enzymatically. Chemical deglycosylation m-ay involve for example, exposure of the GDP Trap polypeptide to the compound trifluoronethanesulfonic acid, or an vqd lent compoid. This treatment results in the cleavage of mostor all sugars except the linking sugar N acetylglucosamine or N -acetyl gal actosamine), while leaving the amino acid sequence intact. Chemical deglycosyiaton is funher described by Hakimuddin et al. (197 Arch. Bioctebm Biophys. 259:2 and by Edge et al (1931) Anal iochem. 1 :3 Enzymatic cleavage of carbohydrate mainties on G(DF Trap pclypeptfides can be achieved by the use of a variety of endo- and exo-Lycosidases as described by Thotakura et at ( 1987) Meth. Enzymol. 138:350. The sequence ofa GDF Trap polypeptide may be adjusted, as appropriate, depecngrm on the 30 type of expression system used, as mammal ian, yeast, insect and plant cells may all introduce differing glycosylation pattems that can be affected by the amino acid sequence of the peptide. In general, GDP Trap polypepides for use in humans will be expressed in a marmalian cell line that provides proper giycosyladon, such as HEK293 or CI) cell lines, although other mammalian expression cell lines are expected to be useful as well 'liis disclosure further contenlates a method of generatig variants, particularly sets of combinatorial variants of a GDF Trap polypeptide cinclding, optionally, truncation variants; pools of combinatorial mutants are especially usefid Or identifying GDF Trap sequences. The purpose of screening such combinatorial libraries may be to generate for example, GDF Trap polypeptide variants which have altered properties, such as aered pharnacok netes, or alered ligand binding. A variety of screenig assays are provided belowand such assays may be used to evaluate variants. For example, a GDF Trap polypeptide vaant may be screened fOr the abihty to bind to an ActRIB pol peptide, to prevem binding of an ActRilB ligand to an ActRilB polypepade or to interfere with signaling caused by an ActIIB gliand The activity of a ODFTrap polypeptide or its variants may also be tested in a cell based or in vivo assay For example, the effect of a GDF Trap polypeptide variant on the expressionof genes involved in hematopoesis may be assessed. This mav; as needed, he performed in the presence of one or more recombinant ActRIIB ligand proteins (e, activin), and cells nay be transfected so as to produce a GDP Trap polypeptide and/or variants thereof and optionally, an ActRilBf ligand, Likewise, a GDF Trap polypeptide may be admniistered to a mouse or other animal, and one or more blood measurementssuch as an RBC count, hemoglobin levels, hemnatocrit levels, iron stores, or reticuiocyte count may be assessed using art recognized methods Comnbinatoriailyderived variants can be generated which have a selective potency relative to a reference GDFTrap polypeptide Such variant proteins, when expressed from recombinant DNA construts, can be used in gene therapy protocols Likewise, mutagenesis can gve rise to varilats which have intracelularhalfives dramatically different than the corresponding uunoditied GDF Trap polypeptide, For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other processes which result in destruction of, or otherwise inacmivation of an unmodified GDF Trap polypeptide Such variants, and the genes which encode them can be utilied to alter CDFPTrap 31 polypeptide levels by modulating the half-ife of the G Trap polypeptides. For instance a short halflife can gve rise to more transient biological effects and. when part of an inducible expression system, can allow tighter control of recormbinant GDF Trap polypeptide levels within the el. In an Pe fusion protein, mutations may be made in th'ne fay and/or the Fe portion to alter the hal ife of the protein. In certain emibod imnsthe (GD? Trap polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally presentin the ActRllB polypepudes. Such modifications include, but are not limited to, acetylation, carboxylat ionglycosylation, phoshorylation hpidation. and acylation. As aresult r'GDF Trap polypeptides may contain non-amino acid elements, such as polyethylene glycols lipids p'oly- or mono-saccaride arnd phosphates Effects of such non-amino acid elements on the functionality of a iDF Trap polypeptide may be tested as described herein for other GDF Trap polypeptide variants. When a GDF Trap polypeptide is produced in cells by cleaving a nascent form of the GDF Trap polypeptide, post-translational processing may also be important for correct folding and/or function of the protein. Different cells (such as 01-1 H eLa, MCK, 293, W138 N I -3T3 or H EK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modi Hcatiorn and processing of the GD F Trap polypeptides, In certain aspects GDF Trap polypeptides include fusion proteins having at least a portion of an ActRIIB polypeptide and one or more fusion domains Well known examples of such fusion domains include, but are not limited to. polyhistidine, Glu-Glu. glutathione S transferase (GS') thioredoxn, protein A, protein C. an inmunoglobulin heavy chain constant region (e.g., an Ec) malose binding protein (MBP), or human serum albumin,. A fusion domrnain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful fb isolation oftie fusion proteins by affinity chromatography For the purpose of affinity purification, relevant matrices, for affinity chroinatography, suc as gutathione- amylase-, and nice or cobah- conjugatedresins are used Many of such matrices are available in "kit" fWm, such as the Pharmacia IST purification system and the QIAeapress system (iage usefulfsion partners. As another example, a fusion domain may be selected so as to facilitate detection of the GDF Trap polypeptides. Exarnples of such detection domains include the various fluorescent proteis (ie g FP),s well as "epitope tags. which are usually short peptide 32 sequences for vhich a specific antibody is available, Well known epitope tags for which specife monoclonal antibodies are readily available incIude FLAG influenzavirus haemnagglutmnin (HA), and cmye tags, In sonic eases, the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin which allows Zhe relevant protease to partially digest the fusion prote.is and thereby liberate the recombinant proteins therefrom. The berated proteins can then be isolated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, a GDF Trap poiypeptide is fused with a domain that stabilizes the GDF Trap polypeptide in vitv (a "stabilizer domain). By stabilizing"is meant anything that increases serum halIife. regardless of whether tis is because of decreased destruction, decreased clearance by the kidney, or other pharniacokinetic effect, Fusions with the Fe portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties, Other types of fusion domains that may be selected include multimerizing (eg dimerizing, tetrameizing) domains and functional domains (that confer an addiOnaI biological function, such as further increasing red blood cell levels) As a specific example, the present invention provides GDF Trap that is an ActRIlB Fc fusion protein which comprises an extraceular (e g, Iigand-binding) domain of ActRUB polypeptide fised to an Fe domain. The sequence of an exemplary Fe domain is shown below (SEQ ID NO: 6) THTCPPORAPELLGGPSVFLpPPKRIDTLInSRTPEVTCVVV(A)VSHEDPEVKENWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAK GQPREPQVYT L PPSREEMTKNQVSLTCLVKG EYPS DIAVEE SNGQPENNYKTT P PVLDSDG P FLY SKLTV DKSRWQQSNV SCSVMHE ALI(A) HYTQKS£5LSPGKa Optionallythe Fe domain has one or more mutations at residues such as Asp265, lysine 3223 and Asn-434. In certain cases, the mutant FR domain having one or more of these mutations (eg, Asp265 mutation has reduced ability of binding to the Fe receptor relative to a wildtype Fe domain. In other cases, the mutantFe domain having one or iore of these mutations (eg, Asn-434 mutation) has increased ability of binding to the MIH C class I ) related Fcreceptor (FcRN) relative to a wildtype Fc domain. It is understood that different elements of the fusion proteins may be arranged in any manner that is consistent with the desired functionality. For example, a GDE Trap -33poiypeptide may be placed C-terminal to a heterologous domainor, aternativelya heterologous domain may be placed (terminal to a O Trap polypeptide. 'The GDFE Trap polypeptide domain and the heterologous domain need not be adjacent in a fusion protein. and additional domains or amino acid sequences may be included C- or N-terminal to either domnan or between the domains, in certain embodimAts, a GDF Trap fusion protein comprises an anino acid sequence as set fbrth in the formula A-B-C. The B portion is an N- and C-terminally truncated ActRUB polypeptide consistingof the amino acid sequence corresponding to amin acids 26132 of SEQ ID NO: 26. The. A nd C portions may be independently zero, one or more than one amino acids, and both the A and C portions when present are heterologous to B. The A and/or C portions may be attached to the B portion via a linker sequence. Exemplary liners are include short polypeptide linkers such as 2-10 2-5, 2-4, 2-3 Glycine residues;, such as, ~r example: a (ily-Cly-Gly linker Other suitable linkers are described herein above. In certain embodiments, a CDF Trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a leader sequence, B consists of amino acids 26 32 of SEQ ID NO 26 and C is a polypeptide portion that enhances one or more of /n vivo stability.in vivo haL iifeptake/admiristration tissuelocalization or distribution formation of protein complexes. and/or purification In certain enbodiments, a GDF lrap fusion protein comprises an amino acid sequence as set forth in the formula A-B C, wherein A is a 'PA leader sequence, B consists of amino acids 26-1 32 of SEQ ID NO: 26, and C is an innunogkbulin Fe domain. A preferred CDF Trap fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the ODF Trap polypeptides of the present invention contain one or more rndihcations that are capable of stabilizing the GDF Trap polypeptides. For example such modifications enhance the in viwo half life of the GDF Trap pol ypeptides, enhance circulatory half life of the GDF Trap porypeptides reducing proteoly6c degradation of the GDF Trap polypept des. Such stabilizing modifications include, but are not limited to. fusion proteins (including, for example, fusion proteins comprising an GDF Trap polypeptide and a stabilizer domain), modifications of a giycosylation site (including, for example, addition ofa glycosylation site to a ODF Trap polypeptide> and modifications of carbohydrate moiety (including, tor example, removal of carbohydratemoieties ftrom a COP Trap polypeptide), in tbe case of fusion prteins, a GDF Trap polypeptide is fused to a 34 stabilizer domain such as an i9G molecule (eg, an Fe domain) As used herein, the term "stabilizer domain" not only refs to a fusion domain (eg, Fe)as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous polymer, such as polethyen e glycol In certain embodiments. the present invenion makes available isolated and/or purified Arms of the GDF Trap polypept ides, which are isolated from, or otherwise substantially free of, other proteins" In cer-tain em-bodiments, GDT Trap pol vpeptides {(tnmodified orrmoditiecj of the invention can be produced by a variety of art-known techniques. For example, such GDF Trap polypeptides can be synthesized using standard protein chemistry techniques such as those described in Bodansky Mi Principles of Peptide Synthesis, Springer Verlag; Berlin (1993) and Grant G A. (ed) Synthetic Peptides: A User's Guide W. H Freeman and Company, New York (1992> In addition, automated peptide synthesizers are commercially available (e.g, Advanced Chen-fech Model 39; Miigen/Boseareb 900. Alternatively, the GDF Trap polypeptides fragments or variants thereof may be reconbinantly produced using various expression systems (e g. E. col, Chinese Harmster Ovary (CO) cells, COS cells, baculovirus) as is well known in the art. in a further embodiment, the modified or Unmodified GDF Trap polypeptides may be produced by digestion of recombinantly produced fullength GDF rap polypeptides by using fr exarmple a protease, e g trypsin thermolysin, clhymotrypsin, Pepsin, or paired basic amwno acid converting enzyme (PACE). Computer analysis (Using a cornercialiv available software, e.g, Macector, Omega. PCGene, Molecular Simulaion, Inc.) can be used to identify proteoytic clavage sites Alternatively, such OD Trap polypeptides may be produced fom recombi nanty produced fullength GDF Trap polypeptides such as standard techniques known in the art, such as by chemical cleavage (e g, cyanogen brenide. hydroxylamine) 3, Nucleic Acds Encoding GDF Trap Polypeptides in certain aspects, the invention provides isolated and/or recombinant nucleic acids encoding any of the GDF Trap pol ypeptides disclosed herein, SEQ ID NO- encodes a naturally occuring ActRilB precursor poiypeptide, while SEQ 1D NO: encodes a soluble AcRIIB polypeptide, and SEQ ID NOs: 25, 2 30 and 31 encode soluble GDETraps, The 35 subject nucleic acids may be singlestranded or double stranded. Such nucleic acids may be DNA or RNA molecules These nucleic acids may be used, for example, in methods for making ODF Tap polyppides or as direct therapeutic agents (e g., in a gene therapy approach). In certain aspects, the subject nucleic acids encoding GDF Trap poiypeptides are further understod to include nucleic acids that are variants of SEQ ID NOs' 5 25z 27, 30 and 31 Variant nucleotide sequences include sequences that differ by one or more nudeotide substitutions, additions or deletions, such as alelic variants; and will therefore include codmg sequences that di ter from the nucleotide sequence of the coding sequence designated in SEQ ID NOs: 5, 25, 27, 30 and 31, in certain embodiments, the invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98% 99'' or 100% identical to SEQ ID NO:5 25 27 30 or 3 One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ 11D NO: 5, 25, 27, 0 or 31. and variants of SEQ ID NO; 5. 25, 27, 30 or 3 1 are also within the scope of this invention, In further embodiments, the nucleic acid sequences of the invention can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA ibrary, in other embodiments nucteicacids of the invention also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 5 25. 27 30 or 3 1, complement sequence of SEQ ID NO: 5, 25, 27, 30 or 31, or fragments thereof As discussed above, one of ordinary ski in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. For example one could perfoomthe hybridization at 6.0x sodium chloride/sodium cstrate (SC) at about 45 1C, followed by a wash of 2.) SaSC at 50 % For example, the salt concentration in the wash step can be selected from a low stringency of about 20 x SSC at 50 *C to a high stringency of about 0,2 x SSC at 50 C. In addition the temperature in the wash step can be increased fecYn low stringency conditions at room temperature, about 22 *0 to high stringency condiions at about 65 Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. in one embodiment, the invention provides nuclei 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. 36 Isolated icleic acids which differ from the nucleic acids as set fbrth in SEQ ID NO: 5 25 2 30 or 31 due to d ny in the genetic code are also within the scope of the invention, For example a number of amino acids arc designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example CAU and CAC are synonyms for histidine) may resulting "silent" mutations which do not affect the amino acid sequence Af the protein. in certain cibodiments, the G3DF Trap polypeptide will he encoded by an alenative nucleotide sequence Alternative nucleotide sequences are degenerate with respect to the native ODF Trap enleic acid sequence but still encode fr the same fusion protein. In certain embodiments, the ODE Trap having SEQ ID NOt 26 is encoded by an alternative nucleic acid sequence comprising SEQ ID NO: 30, However it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteinswill exist anong mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about35% of the nucleotides) of the nuleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such ndeotide variations and resulting amino acid polymorphisms are within the scope of bis inventn. In certain embodiments, the recombinant nucleic acids of the invention may be operably linked to one or moreregulatory nucleotide sequences man expression construct Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable reguslatorv sequences are known in the art for a variety of host cells, Typically said one or more regulatory nucleotide sequences may include, but are not limited to, promoter s leader or signal sequences, ribosomal binding sites, ranscrptionstart and termination seuences translational start and termination sequences, and enhancer or activator sequences Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters ruay be either naturally occurring promoters, or hybrid pr that combine elements of more than one pmotern An expression construct may be present in a cell on an episome such as a piasnid, or the expression construct may be inserted in a chroosome. In a preferred enibodiment, the expression vector contains a seletable marker gene to allow the selection of transformed host celIss Selectable marker cenes are well known in the art and wul vary with the host cell used.

ln certain aspects of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a GDF Trap polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are a o and are selected to direct expression of the GID Trap polypeptide. Accordingly, the term regulat ory sequence includes promoters. enhances, and other expression control elements. Exemplary regulatory sequences are described in Goedde; Gene Expression '71chnology: Methods in Enzmo/og, Academic Press San iego A (1990). For instance any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DiNA sequences encoding a GDF Trap polypeptide. Such useful expossion control sequences include for example, the early and late promoters of SV40, let promoter adenovirus or cytomegalovirus imrrediate early promoter RSV promoters the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase. the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3 phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphaase g, 1ho, the promoters of the yeast romating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereofE It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfornied and/or the type of protein desired to be expressed. Moreover, he vector's copy number, the ability to control that copy number and the expression ol any other protein encoded by th-vectorsuch as antibiotic markers, should also be considered. A recombinant nucleic acd of the invention can be produced by ligating the cloned gene, or a portion thereof into a vector suitable for expression in either prokaryotic cells, eukaryoiccelIs (yeast. avian, insect or manmmlian) or both. Expression vehicles fr production of a recombinant GDF Trap polypeptide include plasinids and other vectors. For instance, suitable vectors indude plasmids of the types: pBR322-derived plasmids pEMBL derived plasmids, pEXderived plasrmnds. a e plmismmdsand pu erd plasruids for expression in prokaryotic cells, such as L co/i Some mammalian expression vectors contain both prokaryotic sequences to fachitate the propagation of the vector in bacteria, and one or more eukaryotic transcription unils that 38 are expressed in eukaryotic cels, The pcDNA aramp, peDNAUnoo pRc/CMV pSV2gpt, pSV2neo, pSV2-dhfr pTk2, pRSVneo pMSG. pSVT7, pko-n and pH-g derived vectors are examples of mammalian expression vectors suitable or triansfection of eukaryotic cells, Some of these vectors are modified with senuences from bacterial plasimds, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukarvotie cels Alteratively. derivatives of viruses such as the bovne papilloma virus (BPVI), or Epstemn Barr virus (pH EBc, pR EPderived and p205) can be used for transient expression of proteins in eukaryotie cels, Examples of other viral (including retroviral) expression systems can be found betlow in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are wel known in the art. For other suitable expression systems for both prokaryotic and eukaryonic cells, as wel as general recombinant procedures see Moleculw Clongt A abora tor Manua/, 2nd Ed... ed. by Sainbrook, PritscI and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. some instances, it may bedesirable to express the recombinant polypeptides by the use of a baculovirus expression system, Examples of such baculovirus expression systems include pVL-derved vectors (such as pVLl392, pVL1393 and pVL941), pAcUW-derived vectors (ch as pAcUWI and pBiueBacderived vectors (such as the 1 -gal containing plueBac Ill), In a preferred embodiment. a vector will be designed for production of the subject GDF Trap polypeptides in CIO cellssuch as a Pcnv-$cript vector (Stratugene, La Jolla, Calif pcDNA4 vectors (Invitrogen, Carlshad Calif) and pC-no vectors (Promega, Madison, Wiscj. As wil be apparent the subject gene constructs can be used to cause expression of the subject CD! Trap polypeptides in cells propagated in culture, e g, to produce proteins, including fusion proteins or variant proteins, tor purification, This invention also pertains to a host cell transfectd with a recombinant gene including a coding sequence (eg, SEQ ID NO: 4, 5 25, 27 30 or 3 1) for one or more of the subject ODF Trap polypeptides- The host cel i may be any piokaryotic or eukaryotic cell For example, a GDitrap polypeptide of the invention may be expressed in bacterial els such as !. co/i. insect cels (e g, using a baculovirus expression system), yeast or mammaiiar cells Other suitable host cells are known to those skilled in the art. Accordingly, the present invention further pertains to methods of producing the subIject GDF Trap polypeptides, For example, a host cel transketed with an expression 39 vector encoding a GDF Trap polypeptide can be cuhured under appropriate conditions to allow expression of the D iTrap polypeptide to occur. The DF Trap polypeptide may be secreted and isolated from a mixture of cells and medium containing the GDF Trap polypeptide, Alternatively, the GDF Trap poiypeptide may be retained cytoplasmicaly or in a membrane fraction and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts w Sitable media for cel culture are well known in the art. The subject GDF Trap polypepudes can be isolated from cell culture nediun, host cells, or both using techniques known in the art for punfyig proteins, including ion-xchange chromatography, gel filtration chromatography, ultrailtration, electrophoresis ard immunoaffinity purification with antibodies specific for particular epitopes of the DTrap polypeptides, In a preferred embodiment, the GDF Trap polypeptide is a fusion protein containing a domain which fa ilitates its purification. In another embodiment a fusion gene coding fr a purification leader sequence, such as a poly- His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinamt ODE Trap polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni4 metal resin, The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified GDF 'Trap polypeptde (eg, see Hochul i et al.. (1987) J 4romaograp 41 177; and Janknecht et al, PNM4S USA 88:8972). Techniques for making fusion genes are welt known, Essentially,the j of various DNA fragments coding for different poypeptide sequences is performed in accordaincevith conventional techniques, employing biuntiended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate terrini, filling-in of cohesive ends as appropriate. akaline phosphatase treatment to avoid undesirable joining, and enzymatic tgation, In another embodiment, the fusion gene can be synthesized by conventional lechniques includmg automated DN A synthesizers. Alternativeiv. POR arnplification of gene fragments can be carried out using anchor printers which give rie to complementary oveahlngs between two consecutive gcne fragments which can subsequently be annealed to generate a chirneric gene sequence (see, for example, Curren f)Totocols in Molecuhtr Biologyv eds Ausubel et al., John Wiley & Sons 1992). 40 4. Screening Assays In certain aspects, the presentnv entjon relat to the use of the subject GDF Trap pogYpeptides (eg soluble variant ActRUB polvpeptides) to identify conpouInds (agents) which are agonist or antagonists of ActRUB polyeptides Compounds identified through this screening can be tested to assess their ability to iodulate red blood cellhemoglobin and/or reticulocyte leves in vivo or in vire, These compounds can be tested, for example in animaltods There are numerous approaches to screening for th e agents fr increasing red blood ell or bemoglobin levels by targeting ActRRB signaling I certainembodiments high-throughput screen -ing of compounds can be carried out to identify agents that perturb ActRl-inediated eiets on a selected cell ine ln certain embodinents, the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding ofan ActRilB polypeptide to its binding partner such as an ActRIB ligand (e.g activin, Nodal, GDF8,GDFI I orBMAP)Alteratively the assay can be used to identify compounds that enhance binding of an ActRL1B polypeptide to its binding partner such as an ActRllB ligand. [r a further embodimrnent, the compounds can be identified by their ability to interact with an ActRIB polypeptide. A variety of assay formats will sufie and, in light of the present disclosture, those not expressly described herein Will nevertheless be comprehended by one of ordinary skill in the art. As described herein, the test compounds (agents) of the inventon may be created by any combinatora chemical method. Alternatively, the subject compound may be naturally occurring biomolecules synthesized in nor or in .im Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria. yeast plants or other organisms (eg, natural products), produced ciemically (egssnall molecules, including peptaidomuineics), or produced recombhinandy. Tet compounds contemplated by the present invention include non-peptidyl organic molecules. peptides, polypepzides, peptidonimetics sugars, hormones, and nucleic acid Inolecles in a spediic mnbodimnentthe test agent is asrna organic molecule having a iolecular vehigt ofless than about 2000 Daltons. 41 The test compounds of the invention can be provided as single discrete entteOsr provided in libraries of grea coriplexiy, such as made by combinatorial chemistry These libraries can compri se, for example. aIcohois alkyI halides, ankles, aides, esters, afdehvdes, ethers and other classes of organic conpoumds. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially ini initial screening steps. Optionally the compounds may he optonaly derkatized with other compounds and have derivatizing groups that facilitate isolation of the compounds. Non initing examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polybistidine. magnetic beads, gutathione S transferase (G3ST). photoactivatible crossinkers or any combinations thereof in many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable ii order to maximize the number of compounds surveyed in a given period of time. Assays vhich are performed in celLft-ce systems, such as may be derived with puri led or semi-purifed proteins, are often preferred as "primary" screens in thathey can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound Moreover, the effects of cellulair ticily or bioavailability of the test compound can be generally ignored in then viro systemthe assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alterationof binding affinity between an ActRlB pol ypeptide and its finding partner (e g, an AcR1113 igand), Merely to illustrate, in an exemplary screening assay of the present invention, the con)mpound of interest is contacted wih an isolated and purified ActRIlB polypepide which is ordinarily capable of binding to an ActIDa ligand. as appropriate for the intention of the assay. To the mixture of the compound and ActRiB polypeptide is then added to a composition contahng an ActRi3 ligand Detection and quantification of ActRIIB/ActR 1B ligand complexes provides a means for determiinng the compoind' efficacy at inhibitng (or potentiating) complex formation between the AcORlIB polypeptide and its binding protein. The efficacy of the compound can be assessed by generating dose response curves froni daa obtained. uing vanois concentations of the test crnnpound. Moreover, a control assay can also be perfomed to provide a baseline for conparison. for example. in a control assay isolaed and purified ActRiB igand is added to a compostion containing the ActRIlIB poIypeptide, aind the formation of ActRllB/ActRiiB ligand complex 42 is quantiated in the absence of the test compound It will be understood that, in general, the order in which the reactants may be admixed can be varied, and can be admixed siiutaneously. Moreover, in place of puified proteins, cellular extracts and lysatcs may be used to render a suitable celi-fee assay systerr Complex fomiation between the ActR lB polypeptide and its binding protein may be detected by a variety of techniques, For instance, modulation of the formation of complexes can be quantitated using, fir example, detectably labeled proteins such as radiolabeled (e g 2 p 3S.C or H), fluorescently labeled (eg., FITC),or enzyniatically labeled ActRiB polypeptide or its binding protein, by inrnunoassay, or by photographicc detection. In certain embodiments, the presera invention contemplates the use of fluorescence polariation assays and fluorescence resonance energy transfer (FRET) assays in measuring, citner directly or indirectly the degree of interaction between an ActRI polypeptide and its binding protein. Further other modes of detectionsuch as those based on optical w aveguides (PCT Publication W( 96/26432 and US. Pat. No 5,677:1 96), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodinents of the invention. Moreover, the present invention contemplates the use of an interaction trap assay, also known as the two hybrid assay," for identifying agents that disrupt or potentiate interaction between an ActRilB polypeptide and its binding partner. See tor exampleUS Pat. No 5,283317; Zervos et Al (1993) Cell 72:223 -232; Madura et al. (1993) J Bio Chemni 268;1204612054; Bartel et al (1993) Biotechniques 14:920-924; and wvabuchi et al. (1993) Oncogene 81693 -1696 in a specifc embodiment, the present invention contemplates the use of reverse two hybrid systens to identify compounds (egsmall molecules or peptides) that dissociate interactions bctwee'n an Act HB polypcptidc and its binding protein. See for example.. Vidal and Legrain, (1999) Nucleic Acids Res 27:919 9 Vidal and Legrain, 1999) Trends Biotechnol 17374-81; and MS Pat Nos. 5525,490 5 955,280;and 5,965.368, In certain emdbodimoents; the subject compounds are identihed by their ability to interact with an ActRUB polypeptide The interaction between the compound and the ActRiMB poypeptide nay be covalent or noneovalent. For example, such interaction can be identified at the protein level using in viPj biochemical methods, including photo crosslinkiig radiolabeled igand binding, and anity chronmatography akoby WB et al. 1974, Methods in Enzyinology 46: 1), In ceie cases, the compounds may be screened in a 43 mechanism based assay, such as an assay to detect compounds which bind to an ActRUB poiypeptide; This may include a sofd phase oluid phase binding event, Atemnaively the gene encoding an AcfRiklB polypepti de can be transtected with a reporter system (eg. f galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library preferably by a high throughput screening or with individuaI neibors of the library, Other mechanism based binding assays may be used, for example binding assays which detect changes in free energy Binding assays can be performed with the target ied to a well, bead or chip or captured by an i mmobiiized d resold by capillary electrophoresis. The bound compounds may be detected usually using colorimetric or fluorescence or surface plasmorn resonance Exemplary Therapeutic Uses In certain embodiments, the GDF Trap polypeptides ofthe present invention can be used to increase red blood cell levels in marinas Such s rodents and primates, and particularly human patients In certain embodiments, the present invention provides nmethods of treating or preventing anemia in an individual in need thereof by adrrinistering. to the individual a therapeutically effetive amount of a GD? Trap polypeptide. These methods may be used for therapeutic and pophylactic treatments of mammals, and particularly humans. As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound that, in a swtistical samplereduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample or delays the onset or reduces the seventy 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 anielioration or elimination of' the condition once it has been established, In either case, prevention or treatment may be discerned in ie diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent As shown herein, GD" Trap polypeptides may be used to increase red blood el, hemoglobin or retculocyte levels in healthy individuals, and such GDF Trap polypeptides may be used in selected patient populations. Examples of appropriate patient populations 44 include those with undesirably low red blood cell or hemoglobin levels,,such as patients having an anemia, and those that are at risk fbr developing undesirably low red blood cell or hemoglobin levels, such as those patients that are about to undergo major surgery or other procedures that TWay result in substantial blood loss, Int one einibodimlent, a patit with adequate red blood cell levels is treated with a GDF Trap polypeptide to increase red blood cell levels, and then blood is drawn and stored fr later use in tmansfusions. GDF Trap polypeptides disclosed herein may be used to increase red blood cell levels in patients having an anemia. When observing hemoglobin levels in humans a level of less than normal for the appropriate age and gender category may be indicative of anemia, although individual variations are taken into account. For example, a hemoglobin level of il2 g/di is generally considered the lower limi of normal in the general adult population Potential causes include blood-loss, nutritional deficits medication reaction, various problems with the bone narrow and many diseases More particAarly, anem-da has been associated with a variety of disorders that include, for example, chronic renal failure, uxytlodysplastie syndrome rheumatold arthritis, bone marrow transplantation, Anemia may also be associated with the following conditions solid tumors (e.g breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (eig. chronic lymphoeeleukemia, nonTodgkns and Hodgkins lyrnphomas) tumors of the hernatopoietic system (eg. Seukeia. myelodysplastic syndrome, rnulie myelorna); radiation therapy; chemotherapy (e.g. platinum containing regions); inflamnmatory and autoimmune diseases, including, but not liirni ted to, rheumatoid arthritisother inflammatory arthritides systemic lupus erythematosis (SLE. acute or chronic skin diseases (e.g. psoriasis), infnammatory bowel dWsease (eg. Crohn s disease anddlerative coiitisy; acute or chronic renal disease or failure including idiopathic or congenital conditions; acute or chronic liver disease; acute or chronic feeding: situations where transfusion of red blood cells is not possihe due to patient allo- or auto-antibodies and/or for religious reasons (e.g. somehova Witnesses); infection (e-g. maria, osteomyeitis; hemoglobinopathies, including for examplesicke cell disease. thalassemias; drug use or abuse, e.gakohol misuse; pediatric patients with anemia trou any cause to avoid transfusion; and elderly patiens or patients wih undedying cardiopulmonary disease with anemia who cannot receive tWansfsicons due to concerns about circulatory overload. 45 ODE Trap polypeptides would be appropriate for treating anemias of hypoproliferauve bone marrow. whichare typicaly associated with ittle change irred blood cell (RBC) morpholgy Ilypoproliferative anemias include: 1) anemia of chronic disease, 2) anemia of kidney disease, and 3) anemia associated with Iypometabolie states. In each of these types, endogenous ervthopoietin levels are jnappropri atel law for the degree otfanemia observed. Other hiypoprolIi ferativye anemias include; 4) early-stage iron-deficient anemia, and 5) anemna caused by damage to the bone marrow. In these types, endogenous erythropoietin levels are approprately elevated for the degree of anemia observed. The mosi comimon type is anenia of chronic diseasewhich encompasses inflammation, infection, tissue injury, and conditions such as cancer, and is di stingui shed by both low erythropoietin levels and an inadequate response to erythopoetiri in the bone marrow (Adanson, 2008, Harrison's Principles of Intemal Medicine 17th ed.. McGraw Hill, New York, pp 628-634) Mi any factors can contribute to cancerrelated anemia, Some are associated with the disease process itself and the generation of inflamatory cytokines such as interleukin- inte-frf n-gamna and iumor necrosis factor (Bron et al 2006 Semin Oncol 28(Suppl 8 1-6) Among its effects, inflammation induces the key iron-regulatory peptide hepeidin, thereby inhibiting iron export from macrophages and generalyiiniting iron availability for erythropoiesis (Gans, 2007, J Am Soc Neprol 18:394-400) Blood loss through various routes can also contribute to cancer-related anemia- The prevalence of anemia due to cancer progression varies with cancer type, ranging from 5% in prostate cancer up to 90% in multiple myeloma. Cancer-related anemia has profound consequences for patients. including faigue and reduced quality of life, reduced treatment ettficacyand increased mortalty. Chronic kidney disease is associated with hypoproliferative anemia that varies in severity with te degree of renanpairiert Such anemia is primariay due to inadequate production of erytropoietin and reduced survival of red blood cells, Chronic kidney disease usually proceeds gradually over a period of years or decades to end-stage (Stage-5) disease at which point diaysis or kidney transplantation is required for patient survival Anemia often develops early in this process and worsens as disease progresses. The clinical consequences of anenia of kidney disease are well-documented and include development of left ventricular hypertrophy, ipaimed cognitive finctione, rduced quality of lif and alered inn)mune unetion (Levin et al 19999 Am J Kidney Dis 27347-354; Nissenison, 1992, Am J 46 Kidney Dis 20(Suppi 1):21-24; Revicki c at, 1995, Am J Kidney Dis 25:548-554; Gafher et aL, 1994, Kidney It 45:224:23 1 As demonstrated by the Applicants in a mouse model of chronic kidney disease (see Example below), a GDF TFrap polypeptide can be used to treat anemia of kidney disease, Many conditions resulting in a hypornetabolic rate can produce a mild-to-moderate hypoproliferative anemia, Among such conditional are endocrine deficiency states, For example, anemia can occur in Addison's disease, hypothyroidism, hyperparathyroidism, or males who are castrated or treated with estrogen. Mildo-moderate anemia can also occur w i th reduced dietary irtakie of protein, a condition particularly prevalent in thie elderly. Finally, anemia can develop in patients with chronic liver disease arising fromt nearly any cause (Adanson, 2008, l arison's Principles of hiternal Medicine, 1 7th ed, McGraw Hill, New York, pp 628-634), Anemia resulting from acute blood loss of sufficient volume, such as from trauma or postpartum hemorrhage, is known as acute post-hernorrhagic anernia. Acute blood loss initially causes hypovolemia without anemia since there is proportional depletion of RBCs along with other blood constitute t.s However, hypovolet ia will rapidly trigger physiologic mechanisms that shift fluid from the extravascular to thre vascular compartment, which results in hemodilution and anemia. If chronic, blood loss gradually depletes body iron stores and eventually leads to iron deficiency. As demonstrated by the Applicants in a mouse nodel (see Example below), a GDF Trap polypeptide can be used to speed recovery from anemia of acute blood loss, Iron-deficiency anemia is the final stage in a graded progression of increasing iron deficiency which includes negative iron balance and iron-deficient erythropoiesis as intermediate stages, Iron deficiency can result from increased iron demand, decreased iron intake, or increased iron loss, as exemplified in conditions such as pregnancy, inadequate diet, intestinal malabsorption, acute or chronic infiammation, and acute or chronic blood loss, With nild-to-moderate anemia of this type, the bone narrow remass hypoproheirat ve, and R BC morphology is largely normal; however, even mild anemia can result in some microcytic hypochronic RBCs, and the transition to severe iron-deficient anemia iS accompanied by hyperproliferation of the bone marow and increasingly y prevalent microcytic and hypochromic RBCs (Adamson, 2008, Harrison's Principles of Interma Medicine, 17th ed.; McGraw Hill, New York, pp 628-634). Appropriate therapy for iron-deficiency anemia 47 depends ctt its cause and severity; xith oralron preparations.parenteral iron fornnuations, and RBC transfusion as major convenional options An GDF Trap polypeptide could be used to treat chronicron-deficiency anemias alone or in combination with conventional therapeutic approaches. particularly to treat anemias of mndtifactorial origin Iypoproliferative anemias can result from primary dysfunction or failure of the bone marrow, instead ofvdysfuncion secondary to infanmmaton, infection, or cancer progression Prominent examples would be rnyelosuppression caused by cancer chemotherapeutic drugs or cancer radiation therapy. A broad review of clinical trias found that mil anemia can occur in 100% of patients after chemotherapy, while more severe anemia can occur in up to 80% of such patients (Groopman et al 1999, 3 Nad Cancer Inst 9;:16161634) Myelosuppressive drugs include: 1) aikylaTg agents such as nitrogen mustards (e.g., melphalan) and nitrosoureas (eggstreptozocin); 2) anti metabolites such as folk acid antagonists (e.g, methotrexate) purine analogs (e g. thioguanine), and pyrimnidine analogs (e.g, gemcitabine); 3) cytotoxic antibotics such as anh.racyclins (eg. doxorubicin); 4) kinase inhibitors (e.g. gefitinib); 5) mitotic inhibitors sud as taxanes (e. paclitaxel) and vea alkaloids (eg. vinorelbine); 6) monoclo na antibodies (e.g. rituximab); and 7) topoisonerase inhibiors (egtopotecan and etoposide). As demonstrated in a mouse model of chemotherapynduced anemia (see Example below), a ODF Trap polypeptide can be used to treat anemia caused by chemotherapeutic agents and/or radiation therapy, OOF Trap potlypeptides would also be appropriate for treating anemias of disordered RBC maturation, which are characterized n part by undersized (ricrocytic, oversized (macrocytic). niisshapen or abnormally colored (hypochromic) RBCs. Patients may be treated with a dosing regimen intended to restore the patient to a target hemoglobin level usually between about 10 gdi and about 12.5 g/dl, and typically about 1 I g/dl (se alsoJobs el al, (2000)Nephroi Dial Transplant 1,5. 15- 19) although lower target levels may cause fewer cardiovascular side effects. Alternatively, hematocrit levels (percentage of the voline of a blood sample occupied by the cells) can be used as a measure for the condition of red bood cells. -leinaocrit levels for healthy individuals range liom 41 to 5 1% for ad uh males and om 35 to 45% for adult females. target hematocrit levels are usually around 30-33% Moreover, hemoglobin/hematoeril levels vary frimn person to person Thus, optimally. the target hemoglobin/hematoerit level can be individualized for eaich patient, 48 The rapid efIet on red blood cellevels of the GJDF Trap polypeptides disclosed herein indicate that these agents act by a different mechanism than Epo. Accordingly, these antagonists maybe useful for increasing red blood cell and hemoglobin levels in patients that do not respond well to Epo. For example, a GDP Trap polypeptide may be beneficial fora patient in which administration of a normal to increased (W00 2Wkg/week) dose of Epo does not result in the increase of hemolobin level np to the target level. Patients with an inadequate Epo response are fbon1 fbr all types of anemia. but higher numbers ofon responders have been observed particularly frequently in patients with cancers and patients with end-stage renal disease. An inadequate response to Epo can be either constittitive (ie, observed upon the first treatment with Epo) or acquired (eg observed upon repeated treatment with Epo). The GDIF Trap go1ypeptides may also be used to treat patients that are susceptible to adverse effects of Epo The primary adverse effects of po are an excessivincrease in the hematocrit or hemoglobin levels and polycytheni evated hematocrit levels can lead to hypertension (more particularly aggraati of hypemtensir) and vascular thrombostOther adverse effects of Epo which have been reported, some of whieh related to hypertension, are headaches, inrduenza- like syndrome, obstruction of shunts, nyocardiai infarctions and Cerebral convulsions duec to thromibosis, hypertensive encephalopathy, and red cell blood cell appiasia (Singibarti, (1994)3 Clin Investig 72(isuppl 6), S36&S43~ HorI et aL (2000) Nephrol Dial Transplant 4$(suppl 451 -56; Delaity e al, (1997) Neurology 49 6S66-9; Bunn (2002) N Engl J Med 346(7), 522-523), (DF traps may also be used in combtnation with Epo and other agents that activate the erythropoietin pathway. In somc instances, this may permit lower dosing of e'ach drug in the combination In certain embodiments, the preserit invention provides methods for managing a patient that has been treated with, or is a candidateobe treated with, a GDF T' ap polypeptide by measuring one or mnore hematiogic parameters in the patient. The hematologic parameters may be used to evaluate appropriate dosing for a patent who is a candidate to be treated with a GDF Trap polypeptide to monitor the hematologic parameters durirnng treatment with a ODE Trap polypeptide, to evauate whether to adjust the dosage during treatment with a GDF Trap polypeptide, and/of to evaluate an appropriate maintenance dose of a GDF Trap polypeptide, if one or more of the hematologic parameters 49 are outside the nomal level, dosing with a GDF Trap polypeptide may be reduced, delayed or terminated. Sematologic parameters that may be measured in accordance with the methods provided herein include; for example, red blood cell levels, blood pressure, iron stores, and other agents found in bodily fluids that correlate with increased red blood cell levels, using art recognized :methods, Such parameters may be determined using a blood sample from a patient. Increases in red blood cell levels, neinoglobin levels, and/or hematocrit levels may cause increases in blood pressure. in one embodiment, if one or nore hematologic parameters are outside the normal range, or on the high side of normal, in a patient who is a candidate to be treated with a DEF Trap polypeptide then onset of administration of the GDP Trap polypeptide may be delayed unti the hematologic parameters have returned to a normal or acceptable level either naturally or via therapeutic intervention, For example, if a candidate patient is hypertensive or prehypertensive then the patient may be treated with a blood pressure lowering agents order to reduce the patient's blood pre , Any blood pressure lower agent appropriate fbr the individual patient 's condition may be used including, for example> diuretics, adrenergicinhibitors (ineiuding alpha blockers and beta blockers) vasodlators, calcium channel blockers angitensi n-converting ezyine (ACE)inhibitor or angiotensin 11 receptor blockerslood pressure may alternatively be treated using a diet and exercise regimen. Similarly, if a candidate patient has iron stores that are lower than normal, or on the low side of nonnal, then the patient may be treated with an appropriate regimen of diet and/or iron supplements until l the patient s iron stores have returned to a normal or acceptable level. For patients having higher than normal re blood cell levels arid/or hemoglobin levels, then administration of the D Trap polypeptide may be delayed until the levels have returned to a normal or acceptable level, In certain embodi ments, if one or nore hematologic parameters are outside the normal range, or on the high side of normal, in a panht who is a candidate to be treated with a ODF Trap polypeptide then the onsel of administration may be not be delayed, However, the dosage amount or frequency of dosing of the GDF Trap polypeptide rmay beset at an amount that would reduce the risk of an unacceptable increase in the hematologic parameters arising upon adnms tratiort of the 0D T) rap pol ypeptide. Altermativlytherapeutia regimen may he developed for the patient that combines a GDF Trap polypeptide with a 50 therapeutic agent that addresses the undesirable level of the hematologic parameter For example, if the patient has elected blood pressure then a therapeutic regimen involving administration of a GDFrap polypeptide and a blood pressure lowering agent may be designed. For a patient having lower than desired iron store, a therapeutic regimen of a ODE Trap polypeptide and iron supplementation may be developed, In one embodiment, baseline parameter(s) for one or more hematologic parameters may be established for a patient who is a candidate to be treated with a GD[ Trap polypeptide and an appropriate dosing renimen establish for that patient based on the baseline values) Altematvely, established baseline parameters based on a patient's medical history could be used to hdtorm an appropriate GDF Trap polypepide dosing regimen for a patient For example, if a healthy patient has an established baseine blood pressure reading that is above the defined normal range it may not be necessary to bring the patient's blood pressure into the range that is consIdered normal for the general population prior to treatment with the GDF Trap polypeptide. A patient's baseline values for one or more hematologic parameters prior to treatment with a ODF Trap polypeptide may also be used as the relevant comparative values for monitorng any changes to the hematologic parameters during treatment with the GDF Trap polypeptide. in certain embodiments one or more hermatologic parameters are measured in patients who are being treated with a GE Trap polypeptide The hematologic parameters may be used to monitor the patient during treatment and permit adjustment or termination of the dosing with the G DI trap polypeptide or add itionaI dosing with another therapeutic agent For example. if administration of a ODE Trap polypeptide results in an increase in blood pressure, red blood cell level 4 or hemoglobin level, or a reduction in iron stores then the dose of the GDF Trap pol peptide may be reduced in amount or frequency in order to decrease the effects of the GDF 'Trap polypeptide on the one or more hemaaoogic parameters. It administration or a GDF Trap polypeptide results in a change in one or more hematologic parameters that is adverse to the patient, then the dosing of the ODE Trap polypeptide may be terminated either temporarily, until the hematologtc parameter(s) return to an acceptable level or permanently Similarly, if one or more hematologic parameters are not brought within an acceptable range after reducing the dose or frequency of admirStration of the GDF Trap polypeptide then the dosing may he terminated, As an altematve, or in addhion to reducing or terminating the dosing with the ODF Trap polypeptide, the patient may be dosed 5 1 with an additional therapeutic agent that addresses the undesirable level in the hematologic parameters) such as, for example a blood pressure lowering agent or an iron supplement; For example if a patient being treated with a GDF Trap polypeptide has elevated blood pressure, then dosing with the GDF Trap polypeptide may continue at the saie level and a blood pressure lowering agert is added to the treatment regimen dosing with the GDF Trap polypeptide may be reduce (e g., in amount and/or frequency) and a blood pressure lowering agent is added to the treatment regimen, or dosing with the ODE Trap polypeptide may be tetmninated and the patient may he treated with a Wood pressure lowering agent. In certain embodiments, patients being treated with a GDF Trap polypeptide, or candidate patients to be treated with a (DF Trap polypeptide, are patients in need of muscle growth such as patients suffering firom. or at risk of developing, a neuronuscular disorder or xusculogenerative disorder. For example, patients or candidate patients may he suffering from, or at risk for developing., Lou GeNrigs disease (ALS)cancer anorexia-cachexia syndrome, rnscular dystrophy, rnuscle atrophy.congestive obstructive pulmonary disease (and muscle wasting associated with COPD), muscle wasting syndrome; sarcopenia, or cachexia, Muscular dystrophy refers to a group of degenerativye muscle diseases characterized by gradual weakeningand deteriorwtn of al muscles and sometimes the heart and respinry nscles. Exemplary muscular dystrophies that can be treated with a regirnen including the subject GDE Trap polypeptides indude- Duchen ne Muscular Dystrophy (DAD), Becker MAvuscular Dystrophy (BM D), Emery-Dreifuss Mnu scular Dystro phy (E DMD), Li mb-Girdle Nuscular Dystrophy (LND) Faci scapukhumeral Muscular Dystrophy (FSH or SHD) s (ao known as Landouzy-Dejerine) Myotonic Dystrophy (MMD) (also known as Steinerts Disease), Oculopharyngeal Museuar Dystrophy (OPMD) Distal Muscular Dystrophy (DD Congenital Muscular Dystrophy (CMD) 6, Pharmaceutical Compostions In certain embodiments compounds (e, GDP Trap polypeptides) of the present invention are formulated with a pharmaceutca ly acceptable carrier. For example, a GDF Trap pol ypepYtide can be administered alone or as a component of a phannaceutical S formulion therapeutict composition). The subject compounds may be formulated for adniintration in any convenient ay for use in human or veterinary medicine. 52 in certan embodiments, the therapeutic mehod of the ivention includes administering the composition systemically Olocally as n implant or device. When administered, the therapeutic composition for usc in this invention is, ofIcourse in a pyroger free, physiologically acceptable forrrn lerapeutically useful agents other than the GDE Trap polypeptides which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds (eig, GDF Trap polypeptides) in the methods of the invention, Typically, compounds will be adinistered parenteraliw. Pharmaceutical compositions suitable for parenteral administration may Comprise one or more GDF Trap polypeptideis in conbi nation with one or more pharmaceutically acceptable sterile isotonic aquoous, or nonaqueous solutionsdispersions, suspensions oremulsions, or stenie powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxid ants, buffers. bacteriostats. solutes w'hih render the tormulation isotonic with the blood of the intended recipient orsuspending or thickening agents Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaeutical compositions of the invention inchde water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and thelike) and suitable mixtures thereof vegetable oils, such as olive oil, and injectable organic e as ethyl oeate Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, Further the composition may be encapsulated or injected in a form for delivery to a target tissue sie (eg, bone marrow). in certain embodimentscompositios of the present invention may include a matrix capable of delivering one or more therapeutic compounds (eg, GDF TPrap polypeptides) to a target tissue site (g, bone marrow), providing a structure for the developing tissue and optimally capable of being resorbed into tie body For exinle,) Ithe matrix may provide slow release of the ODE Trap polypeptidesv Such matrices may e formed ofnaterials presently in use for other implanted media plications. Fe choice of natrix nateria is based on biocornpatibilt, tbiodegradabiity mechanical properties, cosmietic appearance and interface properties The particular application of the subject compositions will define the appropriate femmla4 1tfion. Potential maztrices for the compositions may be biodegradable arid chemically defined calcium sulfate, 53 tricalciumphosphate, hydroxyapatite, polyl actie acid and polyanhydrides. Other potential materials are biodegradable and biologically web defined, such as bone or dermal collagen. Further matrices are comprnised of pure proteins or extracellular mrix components Other potential matrices are nontbiodegradable and chemically definedsuch as sintred hydroxyapatite, biogi ass, aluminates, or other ceranres Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapahte or col agen and lrwcci umphosphate. The bioceramics may be altered in composition such as in calcium-aluminate-phosphat and processing to alter pore size, paricle sizeparticle shape, and biodegradability. In certain embodiments methods of the invention can be administered for orally; eig. n the fbrm of capsules, cachets, pis, tablets, lozenges (using a flavored basis usually sucrose and acacia or traga th), powders, granes, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oiirowater or waterin-oil liquid emulsion, or as an elixir or syrup, or as pastiles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mout washes and the like, each containing a predetermined amount of an agent as an active ingredient An agent may also be administered as a bolus, elecmary or Paste. In solid dosage forms for oral administration (capsules, tablets, pills. dragees, powders, granules, and the like), one or more therapeutic compounds of the present invention may be Axed with one or more pharmaceutical acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose sucrose, glucose, nannitol, and/or silicic aid; (2) binders, such as, farexample, carboxymethylcelulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycero (4) disintegrating agents, such as agar-agar, calcium carbonate potato or tapioca starch, alginic acid, cenain silicates, and sodium carbonate; (5) soluon retarding agents, such as parafin; (6) absorption accelerates, such as quaternary ammoniun compounds; (7) eatingg agents such asa Ir example, Cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay: (9) lubricants, such a talc, calcium stearate, magnesimn sterate, solid poyelhylee glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case ofcapsules, tablets and pills, the phanraceutical compositions may also conprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using 54 such excipients as lactose or milk sugars. as well as high molecular weight polyethylene glycols and the like. Liquid dosage forms fior oral adnmstratin include pharmaceutically acceptable emuisions.microemuisions, solutions, suspensions.syrups, and elixirs, In addition to the active ingredient., theluid dosage fors nay contain inert diluents commonly used in the art, such as water or other solvents, slhbilizing agents and emulsifiers, such as ethyi lcohol, isopropyi alcohol, ethyl carbonate, ethyl acetate benzvl alcohol benzyl benzoate propylene glycol I .butylene glycol oils (in particular, cottonseed. ground nut com, gernolive, castor, and sesame oil) glycerol, tetrahydrofuryl alcohol, pyh l giycols and fatty acid esters of sorbitan, and mixtures thereof Besides inert duents, the oral composnions can also include adjuvants such as wetting agents emudsifying and suspending agents',, sweetening, flavorng, coloring, perfuming, and preservative agents. Suspensions, in addition to the active compoundsmay contain suspending agents such as ethoxylated isostearyl alcohols polyoxyethylene sorbol, and sorbitan esters, rnicrocrystallie cellulose, aluminum inetahydroxide, bentonite, agav-agar and ragacanth, and mixtures thereof. The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for examrtple, parTben, chorobutarut phenol sorbic acid, and the like It may alo be desirable to include isotonic agents such as sugars, sodium chloride, and the like into the compositions. In addition, prokoged absorption of the injectable pharmaceutical form may be brought about by the inchision of agents which delay absorption such as aluminum nonostearate and gelatin It is understood that the dosage regimen wi I be determined by the attending physician considering various factors which modifyi of the subject compounds of the invention (e.g. GDF Trap polypeptides) The various factors include, but are not hmited to, the patients red blood cell count hemoglobin level or other diagnostic assessments, the desired target red blood cell count, the patient's age: se, and diet, the severity of any disease that may be contributing to a depressed red blood cell level, time of administration, and other cliniea factors. The addition of other known growth factors to the final composition nay aso aff the dosageProgress can be b ertodic assessment of red blood cell Pronrss b montore and hemoglobin levels, as well as assessments of reticulocyte levels and other indicators of the hematopoieic process. In certain eibodiments. the present invention also prides gene therapy for the in vivo production of GDF Trap polypeptides. Such therapy would achieve its therapeutic effect by introduction of the COF Trap polynucleotide sequences into cells or tissues having the disorders as listed above. Deliver of C DFTrap polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system, Preferred for therapeutic delivery of GDF Trap polynueleotide sequences is the use of targeted iposomes. Various viral vectors which can be utilized fr gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or an RNA virus such as a retrovirus. The retroviral vector may be a derivative of a marine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney munrine leukemia virus (MoMuLV), Harvey ma ie sarcoma virus (HaMuSV) urine mammary tumor virus (MuMTV). and Rons Sarcoma Virus (RSV) A number of additional retroviral. vectors can incorporate multiple genes. Al of these vectors can transfer or incorporate a gene for a selectable marker so that tansduced cells can be identified and generated, Retrovirai vectors can be nade targetspecific by attaching, for example a sugar, a gycoiipid, or a protein. Preferred targeting is accomplished by using an antibody Those of skill in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the (3DF Trap polynucleotide. Alternatively, tissue culture cels can be directly transfected with plasnids encoding the retroviral structural genes gag. pot and env. by conventional calcium phosphate transfection. These tells are hen transfeeted with the vector plasmid containing the genes of merest. The resudlng cells release the reiroviral vector into the culture medium. Another targeted delivery system fior GDF Trap polynuceotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecucomplexes, nanocapsules, microspheres beads, and lIpid-based systems including oil-in-water emulsions Niceles mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, Liposones are areidcal membrane vesdcles which are useful as delivery vehicles i vitn o and in vivo. RNA, DNA and intact visions can be encapsulated within the aquCous 9-6 interior and be dehvered to cells in a biologically active form (see e.g Fralyet al, Trends Biochen. Sci, 6:77, 198 ) Methods for efficient gene transfer using a liposome vehicle, are known in the art, see e.g., Mannino, et a, iotechniues, 6:682, 1988 The composition of the hiposome is usually a combination of phosphoipids usually in combination with steroids. especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteistis of liposomes depend on pH. ionic strength, and the presence o divalent nations, Examples of lipids usefl in iposome product ion include phosphatidyl compounds, such as phosphatidylglycero, phosphatidylcholine phosphatidylserine, phosphatidylethanolamine sphingolpids, eerebrosides, and gangliosides. Mustrative phospholipids include egg phosphatidyl choli ne, dipalmitoylphosphatidycholineand distearoxlphosphatidylcholine The targeting of liposomes is also possible based on, for example, organspecificity, cell-specificity, and organelle-specificity and is known in the art. EXEMPLIFICATION The invention now being generally descibed t will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention and are not intended to limit the invention Example 1. Generation of a GDF Trap. Applicants constructed a GDF Trap as follows. A polypeptide having a modified extracellular domain of ActRilB with greatly reduced activin A binding relative to GDFI 1 and/or nyostatin (as a consequence of a leucine-to-aspartate substitution at position 79 in SEQ iD NO: I) was fused to a human or mouse Fe domain with a minimal inker (three glycine amino acids) in between The constructs are referred to as ActRllB(79D 20-14) lie and ActRllB(L79D 20- 134)-mFc respectively. Alternative fonns with a glutamate rather than an aspartate at position 79 performed smilady (79E). Alternative forms with an alanine rather than a valine at position 226 with respect to SEQ ID NO: 7, below vere also generated and performed equivalently in all respects tested. The asparate at posinon 79 (relative to SEQ ID NO: 1. or position 60 relative to SEQ I) NO: 7) is highlighted in gray 57 below. The vaine at position 226 relative to SEQ ED NO7 is also g h r below, The GDF'Trap ActR"IB(LT9D 20 134)-Fe is shown below as punfied from C0 cell lines (SEQ ID NO: 7). GRG EAETR ECIYYN ANWELERTNQSCiLER CEC EQDKR LHCY ASW RNSSGTIELVKK GCWDDETNCV? DRQECVATE ENPQYYNCCEGNFCNERNTH 1P EAYGPEVTYEPPPT APT'CIITTC PfCPAPELLOG ES RKKDTLKMISRTPEVTfCVVVDLVS RiDEVK FNW.DGVEVEINAKKPREEQYNSTYP .VSVLTVh LODW LN-KEY KSN KAL PPEKTISKAKGTPQYTLPPSR NOVSLTCLVKGIFYSDILAVEWESNO( __NKT __L SKLEL T VOyK SRWQQGN ESCSVN4 FdLiEA LIYTQKSLS LES P0GK The ActRIIB-derived portion of the GDF Trap has an amino acid sequence set forth below (SEQ ID NO: 32), and that portion could be used as a monomer or as a non-Fc fusion protein as a monomer, dimer or greater order complex. GRCEAETREC1YYNANWELERTNQSGLERCEGEQDKRLHCY ASWRNSSGTIE LVNKKGC WtDDFNCYDRQECVATE EN PQVYFCCOGNFONERFTH LPEAGGEVTYE PPPTAPT (SEQ ID NO: 32) The GDF Trap protein was expressed in CH0 cell lines. Three different leader sequences were considered: (i} Honey bee melittin (IBML): MKFLVNVAVI FM VVYISY IYA (SEQ ID NO: 8) (6) Tissue Plasmi nogen Activator (TPA): MDAMKROGLCCVLLLCGAVFVSP (SEQ ID NO 9) (iii) Native: MTAPWVALALLWGSLCAGS (SEQ ID NO: 10), The selected forn employs the TPA leader and has the following unprocessed amino acid sequence: NM DAMKRGLCCVt I.CGAVFVSPGA SGR FA E TR EC YYNANWELER TNQSCLER CE. CGEQDKRLH CYASWRNSSOI IELVKKGiCWDDDFNCY DRQECVATEENPQVYFCCCE GNFCN ER FTHl EPEAGGPEVTY EPPPTA PTGGTHTPPPGAELL PSV FLPE"K EKD L EM SRTPE VTCVVV DVSH EPPEVKENWYV 'DOVEV HN A KTK PR EEQYN STYRVVS V UTVL.HQDWJNGKEYKCKVSNKALIFPVPIEKTISKAKOQPREEVYTLEPEPSREEMTKNQ 58 VSLTCLVKGFYPSDIAVEWESNGQPENN KTTP PVLDSDGS LY SK LTV DKRWOO I)NVFSCSVMHEAjLHNHYfQJKSLJLSK (SEQ HD NO: I I) This polypeptide is coded by the following nucleic acid se ence(SEQ ID NO, 12): A 'Gci MTc GAMGAGG CTrTGCTGTG TGcTGCTGCT GTGGGCA GT TTCGTT CGCCCGVCGC CTCTCGGCGT GGGGASG CTAGACACGGGA GTGCn C. W TACAACCCA ACTGCGACCT GGACCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGl CA AGCGGCTGWA CTWCTACWCC TCCG=A AWWHCG i CWACAA CTQ N ,A AGGGCGCTG OGACGATAC TTCAACTGCT AC WATAGCA GGAGC 21TG GcACTolcG AGAACCCCCA GGTGTACTIC TGoTGe'rGTG AAGGCAACIT ' C 'CTiT ATTTGC0AGA GGCOTGGGGG CCGAAGTCA CGTACGACCC ACTCCGACA GCCCCG GTGTGGArC TcCAc&rC CCAGCPTGCC CAGGACTCTA ATCTcGGGG GneMTCAC Tm"rCt T CCCCAAAA CCCAe ACA CSCAT c O"cC C (eTCA c2TGC;TGcT GTGACGTG AGcwcACGAG ACCTCTGGT C AAC TGGTACGTGG CGSCiSGA GGMNAT GCAMNAGAA AG7GGjGA 0AASTE A~AC d ACCGTGTGGT CAGCTCCTC ACCMrCCTGC ACCMGACTVOCGATUG AAGGTACA AGMTCAAGT C.CAACAAA CMCTCCAG TCCCATGA 6AAACATC TC$AAAGCU AACCA C CAGAACCA CACGCTACA MCCaICCCC ATCCCCGCAG AGAACCAGT (AGCCTCACC TGCCTGCTCA AASGC'TCTA TCCAGC .AGCCGCG AGGGGAG CAATGGCAG CCGGAdAnGA ACTACAAGAC ACCT TGC CCGACGGCTC CTTCTTCCTC TATAGCAM.C TCACCGTGGA CAACAGG TC:GACAGG GGAACCTCTT CTCATCTCC GTGATCATC AGCCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGT AAATGA Purifcation codd be achieved by a series of column chrormatograph steps, including, for example, three or more of the following n any order; protein A chromatography, Q sepharose hrormatography, jhenylsepharose chromatography size celusion chromatography, and cation exchange chromatography The purification could be completed wih vira Itration and butfer exchangof a purification scheme, the cclI culture medium is passed or a protein A column, washed in 150 mM Tris/NaC (pH 8,0), then washed in 50( mM Tris/NaCf (pH 8,0) and eluted with 0. i M glycine pH 3.0. The low pH em ate is kept at room temperature for 30 mutes as a viral clearance step The eluales then neutralized and passed over a Q sepharose ion exchange column and washed in 50 mM Tris pH 8.0. 50 mM NaCl, and eluted in 50 mM Tris pH 8.0, with an NaC concentration between 150 mM and 300 mM. The cluate is then changed into $0 mM Iris pH 8.0 1. 1 .1 ammonium sulfate and passed over a phenyl sepharose column, washed, and eluted in 50 mM TiAs pH 8.0 with ammonium sulfate between I SO and 300 mnM. The eluate is dialyzed and filtered for use. Additional GDF Traps (ActRLBbFe fusion proteins modified so as to reduce the ratio i activin A binding relative to myostatin or GDF I ) are described in PCTUS2008/001506 and WO 2006/012627,incorporad by efeic. herein 59 Example 2 Bioassay for GDF-1 and Aivinsmediated signaling, An A-204 Reporter Gene Assay was used to evaluate the effects of ActRilB Fe proteins and GDF Traps on signaling by DE-I nd Activin A, Cell line: -Human Rhabdomyosarcma (derived from muscle). Reporter vector: pGL3.(CAGA)l 2 (Described in Dennier et al .1998, EMBO 17 3091-3100); The CAOA 12 motif is present in TGF-Beta responsive genes ( PA- geney, so this vector Is of general use for factors signaling through Smiad2 and 3 Day 1: Spi A-204 cells into 48-well plate, Day 2: A-204 cells transfected with Og pGL3(CAGA)12 or pGL3(CAGA)12() ug)+ pRLCV (I ug) and Fugene. Day 3: Add factors (diuted into medium- 041 % BSA). Inhibitors need to be preincubated with Factors for I hr before adding to cells. 6 hrs Iater. cells rinsed with PBS! and lyse cells This is followed by a Luciferase assay. In the absence of any inhibitors, Activin A showed 10 fd stinulation of reporter gene expression and an ED50 ~ 2 ng/nl. GDF- 1: 16 fold stimulation, EDSO: 1.5 nghm AciRiiB(20 134) is a potent inhibitor of activin, GDF- and D-l activity in this assay, Variants were tested in this assay as well. Example 3 CDF-1 i Inhibition by N-terminal and C-terinal Truncations Variants of ActR IB(20-i 341-hFe with truncations at the N-terminus and/or Ce terminus were gnerated and tested for activity as inhibitors o GDFI- and activin. The activities are shown below (as measured n conditioned media): C-terminal ActR B4h Fe Truncationis: K(n on L)3 DPE I Act v n ActR 20- 34)-hFc 45 22 ctR B11(20- 132)-hFc 87 32 60 ActR11B(20- 131)-Fc 120 44 ActRiilB(20-128)-hFe 130 158 As can be seen, truncations of three (ending with .PPT) six (ending with YEP) or more amino acids at the Cterminus causes a threefold or greater decrease in the activity of the molecule. The truncation of the final 15 amino acids of the ActRilB portion causes a greater loss of actvity (see W02006/012627) Amino temainal truncations were made in the background ofan ActRilB(20-131)-e protein, The activities are shown below (as measured in conditioned media): N-terninal ActRHB-hFc Truncations: I1C50 (igniL) DF- i Kivin ActRllB(20-131 -IFc 183 201 (GRG. )_____ ______ ActRIB(2i-131)~hFc 121 325 (RGE-,) A ct3R 11B(22-131)hFc 71 100 (GEA. -. })____ ____ ActR1l[B(23-13 1)-hFc 50 43 (EAE...) ActRi1B(24-13 )-hFc 9 105 (ART,,,) _ _ _ _ _ _ _ _ _ _ Accordingly, truncations of two, three or fur amino acids from the N-terminus lead to the production of a more active protein than the versions with a full-length extracellular dornain. Additional experiments show that a trncation of five amino acids. ActR 11B(25 131i FEc has actvity equivalent to the untiruncated form and additional deletions at the N terninus continue to degrade-e acity of he protein Therefore. optimal constructs will have a C-terminus ending between amino acid 133-34 of SEQ ID NO: I and an N-tenmnus beginning at amino acids 22-24 of SEQ ID NO: I - An N'erminus corresponding to airino acids 21 or 25 will give activity ihat is similar to the AcBRU1B(20- I 34)-hFc construct These truncations ma also be used in the context ot GDF raps, such as an 1791 or L79E vacant. 61 Exampk 4. ActRIBFe Variants, Cell-based Activtky Activity of ActRI1B-Fe proteins aid (DF Traps was tested n a cell based assay, as described above. Resuts are summered in the tabe below Some vadants were testedin different C treminal truncation constructs As discussed above, truncations of five or fifteen amino acids caused reduction in activity, The GDP Traps (L9D and L79E variants) showed substrial loss of activin binding while retaining aMost wild-type inhibition of GDF- 1 Soluble ActRtR4-Fc binding to GDF I and Activin A: ActR 1-Fe Portio f .ActR lB EL G IDF Inhibition Activin lribiton Variations (cor-esponds to amino Activity Activity acids of SEQ I D NO R64 20v3 +++++ tapprox. 10- NK 1 (approx, 10' M K) A64 20A434 (approx 0 K ) (approx i0 M K,) R64 20-29- - -1h64 K74A 1 20-134 ++++ +++ R64 A24N 20-34 +++ +++.i. R64 A24N 2019 ++- R64 A24N K 74A 20-119 + R64 L9P 20-134 + R64 79P K 7 4A 20134 -+ R64 L79D 20 134 . R64 L'9E 2& 134 ++ R64K 2134 R(64 PI29S Pl3OA 20) 134 +++--I I - ...................... ..... -+ + R64N 20 134 + Poor activity roughlyy I x I10 KI) +Moderate actvhty(roughly ix 1(1K K ) +++ Good (wid4ype) activity (roughly I x IW KO + Greater than wild-type activity Several variants have been assessed for serum hal life in rats AtRF20 34)~Ec has a serum halfife of approxintey 70 hous AcRIB(A24N 20-1 34)4 c has a serm half- ife approximately 100-0 hours. The A24N variant has activity in the cell-based assay (above) and in viv assays (below) that are equivalent to the wildtype molecule Coupled with the longer half-life, this means that over time an A24N varan will give greater efTect 62 per unit of protein than the wl-ype molecule. The A24N variant, and any of the other vari ts tested above, may be combined with the GDP Trap molecules, such as the 199D or L79E variants; Example 5. GDF-11 and Activin A Binding. Binding of certain ActRRB~c proteins and GDF Traps to ligands was tested in a BiaCore assay. The ActRB-F c variants or wild-ype protein were captured onto the system using an anti-hFe antibody Lgands were injeced and flowed over the captured receptor proteins Results are sa nmarzed in the tables. below Ligand binding specificity 11B variants, NGDFII Proten I ( i Koff s)KD ) AcRFIQB2 34) hFc 3 4 6 13c1 4 8,2e 42e ActR.B(A2 4 4 20-134)-h Fe 12I c6 635e-5 5 19e 1 AdRI hi(L79D 20-134)-hiF 6 7e- 5 4.3 9 e 4 6, 5e10 ActRB(L79E 20134)-hFe 2, 7 24e-4 6)0 ActRIIB(R64K 20- 34)1-h hF 6 -5 2 4. 3.56L.. Protein Ron Wi/Ms) Koff (1s) KD (M) ActR3A(20134F 3 69e-5 455 9 35e-l I AcsRllB(A24N 20- 34)-hhc AcdRI1B(L79D 20-134>hFc 85e-5 &3c-4 2. Se-9 A-eMRIIB(LT9E 20-134>hPL A74e- 9e4 24 e- 9 __ ActhdlB(R64K 20I '34)h~I 2 25e5 42 ice 5ie 1e0 Ac RilB0'64K 20-129)-hL ) 74e-4 2 09e-4 2i5' 9 \AR1IB(P1 298 PI30R 2I 8e 5 Se-4 6 ke9 A tRllB(K '4A 20 34%h~ I 2 85 C2.0eS78e Protein Ron UIL~s)TKoffdI/s) K M ActhdllB(20-134-hFc5 94e6 59c-4 2 68e-9 A- RB( A24N 20- -34-h- 3 34e6 3 46-4 -4e 10 ActP IB(L79D 20-134y-hF LOW b nd ng AeRB(L79E 20-134eh4e Low bind ng ActRI13(R412,1 34 hFc 682e6 325e4 476e1 ActRIB(R64K 20-1329)-hf 7 46e6 6 5204 A e-I ActRIIB(P129SP130R 24 &02e6 42 7e4 5 3 c6 63t 134}hFc These data confimn the cell based assay data, demonstrating that the A24N variant retains ligandinding activity that is similar to that of the ActRIlB(20- 4,h,-e molecule, and that the 1.9D or L79E nolecule retains myvosdin and GDF 1 binding but showv markedly decreased (non-quantifiable) binding to Activin A. Other variants have been generated and tested, as reported in W02006/0 2627 (incorporated herein by reference n its entirety) see eg, pp. 5940 using ligands coupled to the device and Howing receptor over the coupled ligands. Notably, K74 K74F, K741 (and presumably other hydrophobic substitutions at K74, such as K74Q) and D801, cause a decrease in the ratio of Actvin A binding to GDI binding relative to the wild-type K74 molecule. A table of data with respect to these variants is reproduced below; Soluble ActRiIB Fe variants binding to GOFI I and Ativin A (BiaCore assay) ActRIIBAcAD l WT (64A) K D= I KD 2 6e7 KM WI (64R) na KD 8 e-8M + tI KD2( ... +++) ............. ++++) E.37A ** R40A D54A K55A ++ 1.* R56A * * K74A KD435e NA KDWU3 9M K74Y * K 41 K74F* E79A * D80K *___ D80R *- * D80N . .. .. * * 64 S F82A I+ * No observed binding <'I /5 WT binding 1/2 WTbinding WT < 2x increased binding A-- ~5 inreqasedbinding ++++ 1 Ox increased binding +- 41 increased binding Example 6. ActRIBAFe Stimulates Erythropoiesis in NoniHurman Primates ActRIlB(20(-34)-4F (IgG 1) was administered once a week for I month to male and female cynomolus monkeys by subcutaneous injectionForty-eight cynoigus monkeys (24/sex) were assigned to one of four treatment groups (6 annualssex/group) and were administered subcutaneous injections of either vehicle or ActRIIB-hFc at 3, 10 or 30 m/kg once weely for 4 weeks (total of5 doses). Parameters evaluated incIuded general cinical pathology (hematology.dinical chemistry coagulation, and urinalysis)ActRB-hFe caused statistically Sigrificant elevated mean absolute retictlocyte values by day 15 in treated arnmals, By day 36. .ActRIIB-TFc caused several hematological changes, including elevated mean absohue reticulocyte and red blood cell distribution width values and lower mean corpuscudar hemoglobin conceiration All treated groups and both sexes were affected. These effects are consistent with a positive effect of ActRila-hPc on the release of miniature reticuloeyes from the bone marrow This effect was reversed after drug was washed out of the treated animals (by study day 56), Accordingly, we conclude that ActARM c-h sti n I ates erythropoiesis. Example 7. ActRB-mFe Promotes Aspects of Erythropoesis in Mice by Stimulation of Splenic Erythropoetie Activities ) in this study the effects of the in i-ko administration of ActR 11B(20-134)-n Fe on the frequency ot henatopoietic progenitors in bone marrow and spleen was aamyzed. One group of 57B36 mice was injected with PBS as a control and a second group of mice administered two doses of ActRlIB-mEFc at 10 mg/kg and both groups sacrificed after 8 days. Peripheral blood was used to perform complete blood counts and femurs and spleens were 5 used to perform i vitr clonogenic assays to assess the lymphoid, erythoid and myloid 65 progenitor cell cogent in each organ. In the brief time frame of this study no significant changes were seen in red blood cell, hemioglobin or white blood cell levels in treated mice. In the fenurs there was no difference in the nucleated cell numbers or progenitor content between the control and treated grups thespleens, the compound treated group experienced a statisticasignificant increase in the mature erythroid progenitor (CFIlE) colony number per dish, frequency and total progenitor number per spleen. In addition, and increase was seen in the number of myeloid (CFUGNM mature eryThroid (BFU-E) and total progenitor number per spleen. Animals; Sixteen C5BU6 female mice 68 weeks of age were used in the study. Eight mice were injected subcutaneously with test compound ActRfIB-mFc at days I and 3 at a dose of 10 mg/kg and eight mice were injected subcutaneously with vehicle control, phosphate buffered saline(B) at a volume of 100 pl.. per mouse. All mice were sacrificed 8 days after first injection in accordance with the relevant Animal Care Guidelines. Peripheral blood (P1B) samples from individual animals were collected by cardiac puncture and used for complete blood counts and differential (CBC/Diff). Femurs and spleens were harvested from each mouse. Tests performed. CBC/Diff Counts PB from each mouse was collected via cardiac puncture arid placed into the appropriate microtainer tubes. Samples were sent to CLV for analysis on a Cellyn3500 counter Clonogenie Assays C lonogeric progenitors of the niyeloid erythroid and lymphoid lineages were assessed using the iniro methylcellulosedbased media systems described below, Mv aturc Erythroid Progenitors: Clonogenic progenitors of the mature erythroid (CFU-E) lineages were cultured in Met hoCultvl 3334, a niethylcellulose-based mediurn containing recombinant human (rh) Erythlropowetir ( U/n L 66 Lyrnphoid P rogenitors: Clonogenic progenitors of the lynphoid (CF (pre-B) lineage were cultured in MethoCult@ 3630, a methylcellulose-based medium containinh Interleukin 7 (10 Lggont) Myeloid and Immature Erythroid Progenitors: Clonogenic progenitors of the granulocyte-monocyte (CFU--GMI, erythroid (BFU-E) and nmiltipotential (CFUGEMM) lineages were cultured in MethoCtulTM 3434. a methylee ose -based medium containing recommant marine (m) Stem CeIFactor (50 ng/m) rh irnTerleukin 6 (10 ng/mL}, rm interleukin 3 (10 ngn L) and rh Erythropoietin (3 Methods: Mouse femurs and spleens were processed by standard protocols Briefly, bone narrOw Was obtained by flushing the fAmoral cavity with Iscove's Modified Dulbecco t s Media containing 2% fetal bovine serum (IMDM 2% FBS) using a 21 gauge needle and I cc syringe Spleen cells were obtained by crushing spleens through a 70 IM filter and rinsing the filter with IMDM 2% FBS. Nucleated cell counts in 3% glacial acetic acid were then performed on the single cells suspensions usi a Neubaer counting chamber so that the total cells per organ could be calculated. To remove contaminating red blood cels, total spleen cells were then diluted with 3 times the volume of ammonium chloride lysis buffer andincubated on ice 10 minutes. The cells were then washed and resuspended in IMDM 2% FBS and a second cell count were performed to determine the cell concentration of cells after lysis. Cell stocks we re rnade and added to each methylcelose-based media formulation to obtain the opt inaI p lai ng concentrations tor eachttssue in each media formulal t ion. Bone m1 a rrow' c ellIS we r e. nayted at i I e0elc I s pe r d sbA if1 n IethoCulI tT M 3 3 34 to ass ess m a turi e erythroid progenitors. 2x 10. cells per dish in MethoCuhaTM 3630 to assess lyinphoid progenitors and 3104 cells per dish in MethoCultTM 3434 to assess immature eryhbroid and m yeloid progenitors. Spleen cells were plated at 4x I Ocells per dish in MethoCuitTM 3334 to assess mature erythroid progenitors. 4x10 edl per dish in MeoulRTNM 3630 to assess lymphoid progenitors and 2 05 cells per dish in MethoCuhTM 3434 to assess immature erytIhroid and mycioid progenitors. Cultures plated in ripicate dishes were incubated at 37*C, 5% CO2 untii coloy enuncmeion and evaluation wxas Iperformed by trained personnel 0 Mature erythroid progenitors were cultured for 2 days, lymphoid progenitors were cultured for 7 days and mature erythroid and myeloid progenitors were cultured for 12 days, Analysis The mean +/- 1 standard deviaionwas calculated for the triplicate cultures oftihe clonogenic assays and for the contMl and treatment groups for all data sets, Frequency of colony forming cells (CFC) in each tissue was calculated as follows; Cells plated per dish Mean oCFC scored per dish Total CFC per femur or spleen was calculated as follows: Total CF scored x nucleated cel] count per f'rnur or spleen (following RBC lysis) Numier of nucleated ells cultured Standard ttests were pertbrmed to assess if there was a differences in the mean number of cells or hematopotetic progenitors between the PBS control mice and compound treated mice. Due to the potential subjectiity of colony enumeration a p value of less than 01 is deemed significant. Mean values (+/- SD) for each group are shown in the tables below. Table Hematologic Parameters STreatnent White Blood Red Blood Cells Hemoglobmn I Hemritocrt Grouj pCells (xi0CL) (xl /) (I'L) (LL) PBS 9 53 -- 1,44 10.3 - LI 160,9 133 0,552 +/- 057 ActRllB-mFc 9,77 4- 1,19 10.8 -1) 162,1 +4,1 0.567 +/- 0.019 68 Table: CFC From Femur and Spleen .. Treatment Total CFC per Total CFC per o p a C per Total CE- per Groop FeurI Spen FemIur\ie PBS8 0 54 4 65 + 27 7 (n=8) AtRlBmFc 8- +/ 9 ~6 + 6*64±+ 23 436 86* (u 8) _ _ _ _ ____ * pretmiinary ardysis indicates p-: 0.05 Treatment of nice w1th ActR IB(20- 34y-mnTc, in the bef tiie frame of this study did not result in sig nican incassin red . od cell or hemoglobin content Hoever, the effect on progenitor cellcontent was notable i the femurs there was no difference in the nucleated cell immbers or progenitor content between the control and treated groups. In the spleens, the compound treated group experienced a statistically sigtficant increase in the nucleated cell number before red blood cell lysis and in the mature erythroid progenitor (CED.1 E) colonynumber per dish, frequency aid total progeirtor number per spleen. In addition increase was seen i the number of myeloid (CFUM immature crythroid (BFUJE) and total progenitnumber per spleen Accordin is expected t over a longer time course, ActRlB( 20-134-inFc treatment may result in elevated red blood cell and hemoglobin content Example 8z A GDF Trap Increases Red Blood CeHI Levels in vivo Twelv-week-old male CS7BL/6NTac mice were assigned to one of two treatment groups (N=:10), Mice were dosed wih either vehicle orwith a variant ActR11B polypeptide (KiD? Trap") [ActRIIB(L9D 20-134yhFej by subcutaneous injection (SC) at 10 mg/kg twice per week for 4 weeks. At study termination whole blood was collected by cardiac puncture. into EDTA containing tubes and analyzed for cell distribution using an H12 hematology analyzer (Abaxis. Ine) Group D tesgn ait------------ Group N Mice section Dose Route Frequency PBS 1 10 C571L/6 0 SC Twicdweek * ,.--.--~-.--.-.-.-.* ~ ..----. *- - -- ------------ ------- _ __________ * 2 10 (:5 713L., GI}Pl'rao It) SC T~ 20i34}-h ] 69 Treatment with the GDF rap did not have a statistically significant efect on the number of White ood cells (W13C) compared to the vehicle controls. Red blood cel (RBC) numbers were increased in the treated group relative to the controls (see table below). Both the heroglobn content (HGB) and hematocrit (tACT) were also increased due to the addjtiona red bHood cells The average width of the red blood cells (RDWe) was higher in the treated animals, indicating an increase in the pool of inmature red blood cells. Therefore treatment with the GDF Trap leads to increases in red blood cells, with no distinguishable effects on white blood cell populations, Hematology Results R RGB HCT RDWC PBS 10,7 0,1 148 i 44, S*04 170±0,1 GDF Trap 12.4 70 u 148.8+ * 18 4* 0A** 0*0* *=p0AS**= pet,01 Example 9: A GDF Trap is Superior to ActRIB-Fe for lcreasing Red Blood Cell Levels In ivo, netee:nweek-old male C57BLJ6NTac mice were randomly assigned to one of three groups, Mice were dosed with vehicle (10 mM Tris Buffered Saline 3S) wild-type ActRlf3(20-i34)-mPFc, or GDF trap ActRhlB(U9D 20-l 34)hPc by subcutaneous injection twice per week for three weeks. Blood was collected cheek bleed at baseline and after three weeks of dosing and analyzed for cell distribution using a hematology analyzer (I-IM2, Abaxis, Inc) Treatment with ActRlIIB-Fc or the GDF trap did not have a significant effect on white blood cell (WBC) numbers compared to vehicle controls. The red blood cell count (RBC). hematocrit (1-CT) and hemoglobin levels were all elevated in GDF Trap treated mice compared to either the controls or the wild-type construct (see table below). Therefore, in a direct comparison, the GDF trap promotes increases in red blood Cells to a significanly greater exent than a wiidtype ActRllB-Fc pFotein. In fact in this experiment, the wildoype ActRllB-Ec protein did not cause a statistically significant increase in red Hood cels. suggesting that longer or higher dosing would be needed to reveal this effect. 70 Hematolog Ruts afer three weeks of dosisug RBC HCT HGB (10 *nl) % g/d1L TBS 1106 ±046 46.78& 9 57 0,7 ActRiIB-mFe 1L,64 A 009 4903 03 16-5 + GDF Trap 13 19 i 0 ** 53 04 18** 8A 03* ** p<:n,01~ Example 10. Generation of a GDF Trap with Truncated ActRIIB Extracellular Domain As described in Example I a GDF Trap referred to as ActRIIB(L79fD 20- 134)-hFe was gernerated by N-terminal fusion of TPA leader to the ActRiIB extracellular domain (residues 20- 134 in SEQ ID NO: I) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO: 1) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) (Figure 3), A nucleotide sequence corresponding to this fusion protein is shown in Figure 4, A GDF Trap with truncated ActR 11B extracelular domain, referred to as AetRilB(L79D 25- 131)-hFe, was generated by N-terminal fusion of TPA leader to truncated extracellular domain (residues 25-131 in SEQ ID NO: 1) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO: 1) and C-terminal fusion of human Fe domain with minimal linker (three glycine residues) (Figure 5), A nucleotide sequence corresponding to this fusion protein is shown in Figure 6. Example 11. Selective Ligand Binding by GDF Trap with Double-Truncated ActRIB Extracelluar Domain The affinity of GDF Traps and other ActRilB-hFe proteins for several ligands was evaluated ie vitro with a fBiacoreTM instrument. Results are summarized in the table below. Kd values were obtained by steady-state affinity fit due to the very rapid association and dissociation of the complex, which prevented accurate determunation of k, and kt 71 Ligand Selectivity of AciRUIIBhFe Variants: Fusion Contruct Activin A Aeivin B GDFI .... (... .- lf .........

) (K di-t -IJ) ... ActR(L79 204 34Lh I L2 36 ActRlB(L79D 2-13)-hFe 1350 73 3 1R3 beB(Le79 2 225(he 2 7A The GDE Trap with a truncated extracellular domain, ActRIB(L79D 25-131)-1 e, equaled or surpassed the ligand selectivity displayed by the longer variant, ActRllB(L79D 20fl 34) hF with pronounced loss of activin A and atvin B bindiy full retention of GDFL I binding compared to AcsRiiih3-Fc counterparts lacking the L79D substitution, Note that truncatin alone (witbout L791) substitution) did not alter selectvity among the ligands displayed here [compare ActR IB( 79 25131 )-hFc with ActRIIB(L79 20-1 34)ybF&L Example 12, Generation of ActRIUB(L791) 25-131)-hFec with Alternative Nudeotide Sequences To generate ActR UB(L79D 25-13 1)-hFec, the human AcIRUIB extraceliular domain with an aspartate substitution at native posinton 79 (SEQ ID NO: 1) and with N-terminal and C-terninal trincations (residues 25-131 i n SEQ ID NO 1) was fUsed N-terminally with a TPA leader sequence instead of the native ActR 111 Pleader and C-terminally with a human Fc domain via a minimal linker (three glycine residues) (Figure 5) One nucleotide sequence encoding this fusion protein is shown in Figure 6 (SEQ ID NO: 27) and an alternative macleotide sequence encoding exactly the sane fusion protein is shown in Figure 9 (SEQ ID NO: 30), This protein was expressed and puiied using the methodology described in Example 1 Example 13. GDF Trap with a Truncated Act-RIIB Extracelludar Domain Increases Proliferation of Erythroid Progenitors in Mice ActRllB(L79D 25-131 -hFc was evaluated to deermine its effect on proliferation of eryihroid progenitors. Male C57BL/6nice ( weeks old) were treated with ActRBIl3(L79D 25-13l)-hFc (10 mg/kg se.: a 6) or vOhc(TBS; n = 6) on Days I and 4 then euthanized on Day 8 for collection of spleens, tibias, femurs, and blood. Cells of the spleen and bone marrow wee isolated, diluted in Iscove's modified Dulbecco's medium containing 5% fetal bovine serum, suspended in specialized nmethyleelasetbased mediurn,and cultured for eithe 2 or 12 days to assess levds of clonogenic progenitors at the coionyforming unit erythro id (CFU-F) and burst forming unit-erythroid (BFULE) stages respectively. Methykcelkdaebased m for BFU F determination (MethoCult M3434, Sem Cel l Technologies) included recombinant rnurine ster cell factor, interieukin-3, and intedeukin 6, which were not present in methylellose medium for CPU-E determination (MethoCult N3334, Stern Cell Technologies), while both media contained erythropoietin. among other constituents. For both BFU- and CFU-E, the number f colonies were determined in duplicate Culture plates derived from each tissue sample, and statistical analysis of the results was based on the number of mice per treatment group. Spleen-derived cultures from mice treated with ActRIIB(L791) 25-31hFe had twice the number of CF- colonies as did corresponding cultures frm control mice (P 005), whereas the number of BFU-E colonies did not differ significantly with treatment in vivo. The number of CFUR or BFE colonies nrom bone marrow cuhures also did not differ significantly with treatment, As expected, increased numbers of CPUF colonies in spleen derived cultures were accompanied by highly significant (P 0 001) changes in red blood cell level (11.6% increase), hemoglobin noncentratIon (12% increase), and hematoerit level (11.6% increase) at euthanasia in mice treated with ActRUB(L79D 25d 31 IhFe compared to controls. These resuls indicate that in vivo administration of a CDF Trap with truncated ActRiiB extracellular domain can stimulate proliferation of erythroid progenitors as part of its overall effect increase red blood cell levels, Example 14. GDF Trap with a Truncated ActRIlIB Extraceliular Domain Offsets Chemotherapy-dticed Anemia in Mice Applicants investigated the effec of ActR liB(L.79D 2.5-13 1)-hFe on erytbropoietic parameters in a nouse rniodel of chemotherapyinduced anemia based on pacliaxcl which inhibits cell division by blocking microtubule polymerizaion. Male C$7B/6 mice (S weeks old) were assigned !,o one of tourtreatments: 1) paclitaxel (25 mg/kg, ip.) 2) ActRUI(ML9.D 25-1 3 Y)hF-e (10 mg/kg. ip) 3) paclitaxel + ActRI B( L79D 25-13 1)-hFe 4) vehie (TBS). 73 Pacitaxel was administered on Day ft while A ctRll3(L79D 25-13 )-hFc or vehicle were administered on Days 0 and 3. Blood samples were collected for CBC analysis from separate cohorts on Days 1, 3 and 5, and results for treatment groups I-3 (above) were expressed as percent difference fron vehicle at a given time point. Attrition due to paclitaxel toxicity was an issue in the pacltaxei only cohort on Day 3 (where n = ); otherwise n=-5 per treatment per time point, Compared to veicle, padlitaxel alone decreased hemoglohin concentration by neady 13% at Day 5, whereas addition of Act RIB(L790 25 131 lyhFc prevented this paelitaxekinduced decline (Figure 11). Similar effects were observed for hematocrit arid R BC levels. In the absence of padliaxel A ctRMBif(IR9D 251i31)-h~e increased hemoglobin concentration by 10% compared to vehicle on Days 3 and 5 (Figure 1I1) Thus, a 3DF Trap with truncated ActRHIB extracellular domain can increase levels of red blood cells sufficienty to offset chemotherapy-induced anemia. Example 15 GfDF Trap with a Truncated ActRI.B Extrace-Rular Domain Reverses Nephrectomy-nduccd Anemia in Mice Applicants investigated the effect of ActRilB(L79D 25-1I3 3-hFc on anemia in a nephrectomized mouse model of chronic kidney disease, Male C57BL6 mic (I1 weeks old) underwent either a sham operation or a unilateral nephrectomy to reduce the capacity for erythropoietin production. Mice were allowed a week for postsurgical recovery and then treated twice-weekly with ActRIB(L79D 25-1 )ihFc (10 mgkg i p; n = 15 per condition) or vehide (TBS; n =15 per condition) for a total of 4 weeks. Blood samples were collected before the onset of dosing and after 4 weeks of treatment. Whereas vehiclereated nephrectomized mice displayed a significant decline in red blood cell number over the 4 week treatment period, treatment with ActRIB(L79D 25-1 1 )-hFc not only prevented the decline utincresed red blood cell lvels 17% (P < 0.001) above baseine (Figure 12), despite reduced renad capacity for erythropoiein production In nephrectomized mice, ActRlliB(1790 .25-131)hb~e also generated significant increases fom baseline in hemoglobin concentration and hemmocrit level and, notablystimuated each of these erythropoiec parameters to approximately the same extent under nephrectomized conditions as under sham-operated conditions (Figure 13). Thus, a GDF ITap with truncated ActRJiB extraceliular domain can increase red blood cell level suffiiently to reverse anemia in a model of chronic kidney disease. 74 Example 16, GDF Trap with a Truncated ActRIlB Extracellular Domain I proves Recovery from Anemia Induced by Blood Loss in Rats Applicants investigated the effect of ActRHB(I79D 25 13hFc on erythropoteic parameters in a rat model of anemia induced by acute blood loss (acute post-hemorrhagic anemia). Male SpragueD-f awley rats (approximately 300 g} received a chronic juguliar catheter at the vendor (Harlan). On Day,. 20% of total blood volume was withdrawn from each rat over a 5--inute period via the catheter under isofturane anesthesia. The volume of blood removed was based on a value for total blood volume calculated according to the following relationship derived by Lee and co--workers (J Nucl Med 25:72-76, 1985) for rats wih body weight greater than 120 g Total blood volume (rl)= R062 x body weight (g)+ 0,0012 An equalvolume of phosphate-buffered saline was replaced via the catheter at the time of blood removal. Rats were treated with ActRUB(L79D 25-131)hFc (10 mg/kgsscn = 5) or vehicle (TBS; n= 5) on Days 0 and 3 Blood samples for CBC analysis were removed via the catheter on Days -I (baseline) 0, 2 4, and 6. Control rats responded to 20% blood loss with a drop of nearly 1%r in red-blood-cell levels by Day 0, These levels remained significantly lower than baseline on Days 2 and 4, and had not recovered fully by Day 6 (Figure 14) Although rats treated with Ac-tRILB(L,79D 25-131)-bFc showed a nearly identical drop in red-bloodu-cell levels after 20% blood loss, these rats then displayed a complete recovery in such levels by Day 2, tilowed by further elevation on Days 4 and 6, which represents a higly significant improvement o ver control levels at the corresponding time points (Figure 14). Similar results were otaned for hemoglobin concentration. These findings demonstrate that a GDE Trap with truncated ActRilB extracellular main can produce a Iester recovery of red blood cel levels front anemia caused by acute hemorihage Example 17. GDF Trap with Truncated ActRIB Extracelhar Domain Increases Levels of Red Blood Cells in Non-Human Primates Two GDF Traps, ActRB(I79D 20-I34yhf and ActRIflB(L79D 251 I31)-hf-c were evaluated fr their ability to stimulate red blood cell production n cyom-olgus monkey. Monkeys were treated subcutaneously with GDF Trap (10 mg/kg 4 nales/4 females or vehicle (n 2 mles/2 females) on Days I and 8-Blood samples were colected on Days 1 75 (pretreatment baseline), 3, S, 15 29, and 44, and were analyzed for red blood cell levels (Figure 15)hematocrit (Figure 16), hemoglobin levels (Figure 17) and reticulocyte levels (Figure 18), \ehicledreated monkeys exhibited decreased levels of red blood cells, hematocnt and hemoglobin at al post-treatment time points, an expected effect of repeated blood sampling. in contrast, treatment with ActRIlB(L79D 20-134)-hFe or AcIRIIB(L79D 2543 )-hFc increased these parameters by the first post-treatment time point (Day 3) and maintained them at substantially elevated levels fAr the duration of the studg (Figures 1 517), importantly retculocyte levels in monkeys treated with ActR iB(l79D 20-34)-hFc or Acf Ii B( 179D 25-131)h1-e were substantially increased at Days 8, M and 29 compared to vehicle (Figure 18). This result demonstrates that GJDF Trap treatment increased production of red blood cell precursors, resulting in elevated red blood celevels Taken together these data demonstrate that truncated GDF Traps. as well as a full length variants, can be used as selectiye antagonists of GDF I and potentially related ligands to increase red blood cell formation La ivo Example I& GDF Trap Derived from ActRilB5 Others have reported an alternate, soluble form of ActRllB (designated ActRUiBS), in which exon 4, including the ActR13 transmembrane domain. has been replaced by a different C-teminal sequence (102007/053775). 'The sequence of native hunan ActRlllB5 without its leader is as follows GRGEATREC I Y NANWLERTNQSGLERQEGEQUKRLHCYASW RN SSGTIELVK KGCN4LDDENCYDRQFECVATEENPQ FCCCEGFN ECN ER FTH LPEAGGPEG £WAST T1 PSGGPEATAAAGDQGSGAL LEGPA HE (SEQ IE) NO: 36) An leucne-aspartate substitution or other acidic substtutions may be perfOrmed at native position 79 (underlined and highlighted) as described to construct the variant ActRHBS(l)79D), which has the llowin ieuence: GRGEAETRECIYYNANWELERTNQSGLERGEGEQDKRLHCYASWRNSSGTINLK KGCWDDFNCYDRQECVATEEN PVY CCCEGNFCNERFTHL EAGGPEGPWAST NIPSGGPFMTAAAGDQGSGAIWICEGPANE (SEQ ID NO; 37) 76 This variant may be connected to human F with a TOGG linker to grenrate a human ActRSlB5(39DY-hFc fusion protein with the follIowing sequence: GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVK KGCWDDDFNCYDRQECVATEN PQVY FCCE GFCNERLT tPEnAGGPEGPWA ST TI PSGGPEATAAAGDQGSGALWCL EGA E&GGGTHTCPPCPA PLVLGGESLE FPPKPKDTLMISRT PEVTCVVVDVSH EDPEVKENWTVDGVEVHNAKTKPREEQY5N STYRVVSVITVHQDWLNGKEOGKVrNKAiPAPIEKT ISKAKGQ PREPQVYTLa PSREEMTKNQVSLTCIVKGFYPSDIA VWESNGQPENNYKTTPPVLDSDGS FFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38) This construct may be expressed in CHO cells, INCORPORATION BY REFERF.NCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individual indicated to be incorporated by reference While specific embodiments of the subject mater have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the daims below. The full scope of the invention should be determined by reference to the claims, along vith their ful I scope of equivalents and the specification, along with such variations, 77

Claims (18)

1. 2. The method of claim 1, wherein the acidic amino acid is selected from the group consisting of: a glutamic acid and an aspartic acid,
2. 3. The method of claim 1 wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 29- 109 of SEQ ID NO: 1, 4, The method of claim n wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 25- 18 of SEQ ID NO: i, The method of claim I. wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 25128 of SEQ ID NO. 1. The method of claim , wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the sequence of amino acids 254 31 of SEQ MD NO: I. The method of claim I, wherein the polypeptide comprises an amino acid sequence that is selected from the group consisting of: SEQ ID NOS,7. 28, 29, 32, 37 and 38. 8, The method of any of claims I to 7, wherein the patient has anemia associated with a kidney disorder.
3. 9. The method of claim 8. wherein the patient has anemia associated with chronic kidney disease, I0 The method of any of claims Ito 7 herein the patient has anemia associated With a chemotherapy treatment. 1, The method of claim 10, wherein the chemotheapv treatment is a taxane. 78 12, he method of any of claims I to 7, wherein the patient has anermia as a consequence of blood koes,
4. 13. A polypeptide comprising the amino acid sequence of SEQ UD NO. 29
5. 14. The polypeptide of claim 13, wherein the polypeptide rther comprises a constant domain ot an imrunogkohud n. IR The po peptide olcdam13, wherein t polypeptide further compriseS an Fe domain.
6. 16. The polypeptide of Claim 13, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 28.
7. 17. A polypeptide comprising the amino acid sequence of SEQ ID NO: 32, 18 The polypeptide of claim 17, wherein the polypeptide further conpses a constant region of an immunoglohdin
8. 19. The polypeptde of claim I where the polypeptide further comprises an -c domain. 2O The polypeptide of claim 17, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO: 7, 21 Ihe polypeptide of daim 17, wherein the portion of the polypeptide derived from ActRilB has an amino acid sequence that consists of the amino acid sequence of SEQ ID NO;29.
9. 22. A polypeptide according to any of claims 13 to 2 1 wherein the po ypeptide further comprises one ot more modified amino acid residues selected from: a gIycosylaed amino acid, a PEGylated aino acid, a faresylated amino acid, an acetylated amino acid a biotinylated amino ui, an amino acid conjugated to a lipid moiety and an amino acid conjugated to an organic derivatzing agent 79 2 3 A fusion protein comprising an amino acid sequence as s forth in the formula A-B C, wherein B? is a polypeptide with an amino acid sequence consisting of the amin4LO acid sequence of SEQ ID NO: 29; wherein A and C are independently zero, one or more than one amino acid; and wherein A and C are heterologous to B.
10. 24. The fusion protein of claim 23 vherin the fusion protein forms a homodimet 25 The fusion protein of claim 23 or 24wherein at least one of A or C comprises a polypeptide portion that enhances one or more of in avo stability, in vivo half life, uptakedadministrai tissue localtzatiorl or distribution, formation of protein complexes, and/or purieai on.
11. 26. The fusion protein of claim 23 or 24, wherein at least one of A or C comprises a constant region derived from an IgG heavy chain. 27 The fusion protein of claim 26 wherein the constant region derived from an lgG heavy chain is an Fc doman., 28, The fusion protein of claims 23, wherein A, C or A and C are fused to B using a linker. 29 The fusion protein of claim 28, wherein the linker comprises Gly-Gy-Gly,
12. 30- A fusion protein of any of claims 23 to 29, wherein the fusion protein includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated anina acid, a famesylated amino acid. an acetylated amino acid a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivating agent. 31 The fuiion protein of' claim 30, wherein 8 contains at least one modified amino acid residue, 32, A phannaceutical preparation comprising a polypeptide if any one of claims I3 to 3 1
13. 33. A cultured cell conprising a nucleic acid that encodes a polypeptide of any of claims 13 to 3 1, 80 34 The cultured cell of claim 33, wherein the cell is a manimalan cell, 35 The cultured cell of claimn 34 herein the cell is a CHO cell. 360 The cultured cell of claim 33 Wherein the nucleic acid hybridizes under stringent hybridization conditions to the nucleicacid sequence selected from the group consisting of. SEQ ID NOS: 12, 25, 27, 30, 31. 33, 34 and 35. 3T The cultured cell of claim 33, wherein the nuclec acid is at least 95% identical to a nucleic acid sequence selected front the group consisting of: SEQ ID NOS: 12, 25, 27, 30,3, 33 34 and 35. 38 The cultured cel of daim 33, wherein the nucleic acid is selected f&om the group consising of: SEQ I N(: 12 25,27 30, 3133, 34 and 35 39 The cultured cell of clain 33, wherein the portion of the nucleic acid that encodes fr ActR 11derived amino acid sequence, tinker sequence and/or immunoglobulin sequence consists of the nucleic acid sequence selected from the group consisting of SEQ I D NOS t 2, 2 27, 30 1 33 34 and 35,
14. 40. A method for increasing red blood cell levels or treating an anemia in a patient, the method comprising administering to a patient in need thereof an effective amount of a variant ActRB polypeptide, wherein the variant ActR IllB polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids 29 109 of SEQ ID NO: 1, and wherein the variant ActRhlB polypeptide has a ratio of GDPI IActivin finding that is greater than the ratio for the corresponding wild-type AcIRiB polypeptide 41, The method of chrn 40, wherein the variant ActRUB polypeptide comprises an acidic amino acid at the position coesponding to position 79 of SEQ ID NO: 1
15. 42. The method ofcdairn 40, wherein the patent is a human patient.
16. 43. The method ofclairm 40, wherein the variant ActR llB polypeptide has a ratio of dDF I l:Acivin A binding that is greater than the ratio for the corresponding wild type ActRlB polypeptide. 81 44 The method of claio 40, wherein said variant AcR IHB polypeptie comprises an amino acid sequence that is at least 90% identcal to a sequence selected from the group consisting of: (a) a sequence beginning at any of amino acids 22-24 of SEQ 1D NO I and ending at any of amino acids 133-134 of SEQ ID NO: 1; (b) a sequence beginning at any of amino acids 21-29 of SEQ ID NO: and ending at any of amino acids 1 38 134 of SEQ ID NO: (c) a sequence beginning at any of amino acids 21 29 of SEQ ID NO: I and ending at any of amino acids 128I34 of SEQ ID NO: ; (d) a sequence beginning at any of amino acids 2124 of SEQ ID NO; I and ending at any of amino acids 109 134 of SEQ ID NO: I; (e) a sequence beginning at any of amino acids 21F4 of SEQ W0 NO: 1 and ending at any of amino acids 118-134 of SEQ ID NO: 1; 1, a sequence beginning at any of amino acids 21-24 of SEQ [ID NO- 1 and ending at any of amino acids 1281 34 of SEQ ID NO: 1; (g) a sequence beginning at any of amino acids 20-29 of SEQ ID NO:I and ending at any of anino acids 109-133 of SEQ ID NO: I; (h) a sequence beginning at any of amino acids 20-29 of SEQ i) NO: I and ending at any of amino acids 1184 33 of SEQ ID NO: I; (i) a sequence beginning at any of anino acids 20-29 of SEQ ID NO; 1. and ending at any of amino acids 128133 of SEQ ID NO: 1; () a sequence beginning at any of amino acids 20-24 of SEQ ID NO: I and ending at any of amino acids 109133 of SEQ ID NO: 1; (k) a sequence beginning at any of amino acids 20-24 of SEQ ID NO I and ending at any of amino acids I18 133 of SEQ ID NO: 1;() sequence beginning at any of amno acids 20424 of SEQ 1D NO. 1 and ending at any of amino acids 128-I33 of SEQ ID NO: 1; and (m) a sequence beginning at amno acid 20 of SEQ I) NO: 1 and ending at amino acid 134 of SEQ ID NO- I, 45, The method of any of claims 40 to 44, wherein said variant ActR 1B polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a Nnesylated amino acid an acetw ted annno acid, a biotinylated amino acid, anamino acid conjugated to a lipid naoietyand an amino acid conjugated to an organic derivanting agent 46 The met hod of any of clains 40 to 44, wherein thl increase in red blood cell levels is measured as an increase in hemoglobin levels in the blood, 82 47 The method of any of claims 40 to 44, wherein the method causes less than 15% increase in the patients skeletal musle mass 4& [he method of any of claims 40 to 44, wherein said variant AckRlB polypeptide is a fusion protein including, in addition to a variant AcIRIIB polypeptide domain, one or more polypeptide portions that enhance one or more of in vVO stability, in vivo half ifE uptakeadmnistration tissue localization or distribution, formation of protein complexes, and/or purification. 49, The method of any ofclaims 40 to 44, wherein said fusion protein includes a p olypeptide portion selected front the group consisting ofan immunoglobulin Fe domain and a serum albumiiri. 50 The method of daun 49, wherein the fusion protein is a variant ActR IE-c fusion protein comprising an amimo acid sequence selected from the group consisting of: a) an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, l I28, 29, 32, 37 and 38, lb) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, 11 28,29 32 37 and 38, and c) the amino acid sequence of SEQ ID NO: 7 11, 28. 29, 32. 37 and 38 5 , The method of claim 50, wherein the variant ActRIHB-Fc fusion protein has a serum half-life of between 15 and 30 days,
17. 52. The method of claim 50, wherein the variant ActRl1B-Fc fusion protein is adm-inistered to the patient no more frequently than once per week,
18. 53. The method of claim 50, wherein the variant ActRlii3-Fc fusion protein is administered to the patient no more frequerly than once per month, 83