AU2015201033A1 - Activin-ActRIIa antagonists and uses for promoting bone growth - Google Patents

Abstract

ACTIVIN-ACTRIIA ANTAGONISTS AND USES FOR PROMOTING BONE GROWTH In certain aspects, the present invention provides compositions and methods for promoting bone growth and increasing bone density.

Description

ACTIVIN-ACTRIIA ANTAGONISTS AND USES FOR PROMOTING BONE GROWTH RELATED APPLICATIONS 5 The present application is a divisional application of Australian Application No. 2012238197, which is incorporated in its entirety herein by reference. This application claims the benefit of U.S. Provisional Application Nos. 60/739,462, filed November 23, 2005, 60/783,322, filed March 17, 2006, and 60/844,855, filed September 15, 2006, which applications are hereby incorporated 10 by referenced in their entireties. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. 15 Disorders of the bone, ranging from osteoporosis to fractures, represent a set of pathological states for which there are few effective pharmaceutical agents. Treatment instead focuses on physical and behavioral interventions, including immobilization, exercise and changes in diet. It would be beneficial to have therapeutic agents that promote bone growth and increase bone density for the 20 purpose of treating a variety of bone disorders. Bone growth and mineralization are dependent on the activities of two cell types, osteoclasts and osteoblasts, although chondrocytes and cells of the vasculature also participate in critical aspects of these processes. Developmentally, bone formation occurs through two mechanisms, endochondral ossification and 25 intramembranous ossification, with the former responsible for longitudinal bone formation and the later responsible for the formation of topologically flat bones, such as the bones of the skull. Endochondral ossification requires the sequential formation and degradation of cartilaginous structures in the growth plates that serve as templates for the formation of osteoblasts, osteoclasts, the vasculature and 30 subsequent mineralization. During intramembranous ossification, bone is formed - 1 directly in the connective tissues. Both processes require the infiltration of osteoblasts and subsequent matrix deposition. Fractures and other structural disruptions of bone are healed through a process that, at least superficially, resembles the sequence of developmental events 5 of osteogenesis, including the formation of cartilaginous tissue and subsequent - la mineralization. The press of fracture healing can occur in two ways Direct or prheary bone healing occurs without callus formnainndirect or secondary bone healing occurs with a -calls precursor stage Primary healing of fractures involves the rdennation ofmechanical contimity across a closely-et disruption. Under 5 suitable conditions n cells surrounding the disruption show a turn.ellng resorptive response and establish pathways for the penetration or blood vessels and subsequent healing. Secondary healing of bones follows a process of inflammation, sofallus torn-aton. calus mineralisation and callus reIodelinga in the inmation stage, henatoma and haemtorrhage fonnation results from the 10 disruption of periosteal and en-oste a blood vessels at th;e site of injury. Inflammatory cells invade the area, In soft hallus fomaton stage. the cells produce new vessels. fibroblasts, intracellular material and supporng cetis, forming granulation tissue in the space between the nactur am agents, Clinical uuon across the disruption is established by fibrous or cartifagixs tissue (soft calhis) IS Osteoblasts are forned and amdiate the nen tion of soft callus, which is then replaced by lanellar bone and subjected to the normal remodeling processes,. In addition to fractures and oher physical disruptions of bone structureloss of bone mineral content-and bone mass can be caused by a wide variety of cnudtions and may result in significant niedical problems. Changes to bone mass 20 occur in a relatively predictable way over the life of an individuaL Up to about- age 30 bones of both Inen and women grow to mnaxnnal mass through linear growth of th endochondral growth ptes and radial growth Afer about age 30 (for trabecular bone e-g, flat bones such as the v"tbrae and pe Ivis) and age 40 (frz cortcal bone, eo, long hone: fAunl in ihe limbs), slow bone loss occurs in both men and women. 25 In women, a final phase of substantial bone loss also ccurs, probably due to postmenopausatestrogen deficien c During this phase, women may lose an additional 10% of bone mass from the cortical bone and 25% from the trabecular conpartmenit. Whether pro-ressive bone loss resuts in a pathological condition such as osteoporosis depends largely on the initial ban" mass of the individual and 30 whether tere are exacerbating conditions, Bone loss is sometimes characterized as an imbalance in the normal bone rem-deling process Heahy bone is constantly subject to remodeling. Remodeling -2begins with resorption of bone by osteoclasts. The resorted bone is then replaced by new bone tissuewhich is charscterized by collagen brnation by oste'blsts, ad subsequent calcfcation, in nealthy individuals the raes of resorption and formation are balanced, Osteoporosis is a chronic, progressive condition marked by a shift 5 towards resorption. resulting in an overall crease in bone mass and bone mineralization. Osctoporosis in humans is preceded by Clinical osteoperia (bone mineral density that is greater than one standard deviaton but less than 25standard deviations below the mean value fm oruetmsadlt bone)Y Worldwide, approximately 75 million people arc at isk fr oe Thus, methods fAr controlling the balance between osteclast and osteoblast activity can be useful for promoting the lanu >of bactures and other damage to bone as well as the treatment of disorders, such as osteoporosis,associated with loss of bone mass and bone mineralization, with respect to oseoporosis estrogen calcitonin, osteocalcin with. vitanti 15 K, or high doses of dietary calciun are a] used as therapeutic interventnOther therapeutic approches to osteoporosis include bisphosphonates parathyroid hormones c -a.netics. statiris, anabolic steroidsanthanum and strotium salts, and soiun fuoride, Such therapeunics, however are often associated with undesirable side effects, 20 Ts.iisan obetof tlhe present disclosure to proved conositions ar~d me-tho ,dls for prominug bonel growth and nnnleralhzstaoin SUMMARY OF THE INVENTION in part, the disclosure demonstrates that molecules havMg actlvin or ActPia 2$ antagonist activity ("aevin antagonists and "ActRila antagonists") can be used to increase bone density, prmote bone growth> and/or increase bone strength In particular the disclosure demonstrates that a soluble form ofAsaRIL dcts as an inhibitor of activn-ActRlla signaling and promotes increased bone density bone growth, and bone strength in vivo. whilee most pharmaceutical agerts that promote 30 bone growth or inhbit bone loss act as either anti-ctabolic agents (also commonly .referred to as "Catabolic agents") e g bisnhosphonates) or enabolic agents (e g -3 parathyroid hormone, IH when appropriately dosedit the soluble ActRia protein exhibits dual activity, having both catabciic and anabolic eMths, Thus, the dis cLsure establishes that antagoniss of the activin-ActRIa sgnalng pathway may be used to increase bone density and promote hone growth, Wh Ie sohible ActRita 5 may affect bone through a Inechanisa other than activin antagonisn the disclosure nonetheless demonstrates that desirable theraputit. agents nay be selected on the basis ofan activin~ActR1la antagni st acti vity Therefore in certai emnbodmnents, the disclosure provides methods itr usig actish-AcrR Ila antagonisis including; tor example, activi--bInding ActRITa polypepAides ani artivin antibodies, anti-ActRia 10 anttbodies, aetivin- or ActR a-targeted smallnoiecies and abtamers and nucleic acids that decrease expression of activin and ActifTa, treat disorders associated with low bone density or low bone s eength, such as osteoporosis, or to promote bone growth in patients in need thereof, such as in patients having a bone fiatuSrer Additionally the soluble AotiRa polypeptide promoter bone growth without 15 causing a cnsiste'dy measurable increase in muscle mass inl certain aspectsthe disclosure provides polypeptides cnprising a soluble activ~neinndrig ctRlla polypeplide that binds to activin. ActRila p .lypeptides may be formulated as a pharmaceutical preparation comprising the a<ctivin-binding AMtRa polypeptide and a phanmaceuticIly acceptable carrier, Preferably the 20 activin-binding ActiR-a polypeptide binds to activhin with a Ko less than I. mncromolar or less thman 00, 10 or I nanomolar Optionally the activin-binding ActRita polypeptide selected bids activin vesui-ss GDF11 and/or 3DF8 and preferably with a Ka that is at keast 10 fold, 20-f.od or 50-fold lower with respect to activin thlian with respect to GDF and/or G DXF8 While not wishting to be bound to 25 a particular mechanim of action, cx octed that this dgree of selectivity for activin .nhibition over GDF 1/DP8 in ibit acOun ts 3ar the selective effect on bone vithout a consistently measurable effet tn muscle In may embodiments an ActRia powypeptide will be selected for causing less than 15%, less than 10% or less than 5% increase in muscle at doses tha achieve desirable effects on bone. 30 Prerably the composition is at least 95% pure, with respect to other polypeptide cr oponents a, s assessed by size exclusion chromatography and more preferably, the composition is at least 9,% pure An activin-binding ActRila polypeptide for -4use in such a preparation ma be any of thos disclosed herein, such as a polypeptide having an amino acid sequence selected tom SE Q;NOs: 2, 7 or 12 or having an amino acid sequence that is at least 80% 85%, 90%, 95%y 97 or 99% identical to an amino ad sequence selected fromn \Q ID NOs 2 37 12 or 5 13, An activin-binding ActRila polypeptide nay include a fnctinonalxdragment ofa natural ActRita polypeptide, such as one compdrsing at least 10, 20 or 30 anino acids of a sequence seleted from SEQ ID NOs 13 or a sequence of SEQ ID NO 2 lacking the C-terminal 10 to 15 amin o acids (the "tail). A soluble, activm-'binding ActRib polypeptide may include one or more 10 altrations I the amino acid sequence (. g -in igandbinding domain) relative to a naturally occurring ActRita polypepti vam.pies of altered ActRHla polypeptides are provided in. WIO 2006/01 2627 pp. 5960, incorporated by reference herein The altirtion in the amino ai sequence may, for example, alter giycosyltion of the polypeptide when produced in a mammalian, insect or other 1.5 eukaryotic cell or alter proteoiyti cleavage of the polypepide relative to the naturally occurring ActRIa polypeptide An actitbinding Adtia polypeptide may be a fusion protein that has, as one doa in an ActRita polypeptide (e a lia b indirg portion of an ActRita and one or mnr' additional domains that provide a d"sirable property, such as 20 improved pharnnacokinetics, easier puricarirn targeting to particular tissues, etc. For example a domain of a fusion protein ma enhlance one or More of in vivo stability in vivo half life uptakdadministration, tissue localization or distribution. fmadi ofprtin complexes, uutimnerization of the fusion protein, andior f'oratifon of proki ,n purification. An activinabinding Actiia fusion proteinmay include an 25 imnnunoglobulin Fe domain (wildtvpe or mutant) or a serum albunin or other polypeptide portion that provides desirable properties suh as improved pharmacokinetics, improved solubility or proved stability in a preferred embodiment, an ActRIlD- fusion comprises a relatively unstructured linker positioned between the Fe domain and the extracel tarAcWRa domain his 30 unstructured linker nay correspond to the roughly 15 amino acid unstructured region at the C-terminal end ot the extrace.llular domain of ActRla (the tai) or it may be an artif icial sequence of 1, 2, 3, 4 or 5 amino acids or a length of between $ -5 and 15' 20 30 50 or more animno acids that are relatively free of secondary structure, or a mixture of both. A linker may be richin glycine and proline residues and may, for exampie.contain a single sequence of thronineterine and glyJines or repeating sequel rineocfQkv an lyci. nes ( T04 0gor S 4 singlCts or 5 repeats), A fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequenceand a GST fusion. Optionally, a soluble ActRila polypeptide includes one or more modified amino acid residues seletdm from: a glycosylated anino acidn PEGylated amino acid, a farnesylated amoin acid. an acetylated amino acid, a biotinylared amino acid, an amino acid 10 conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent, A pharmaceuticalpreparation may also include one or more additional compounds suoh as a compound that is used to treat a bone disorder Preferably a phann' acentical preparation .3 substantially pyrogen free I genera, it is preferable that an ActRHa protein be expressed in a mammalian eei line that 15 niedties suitably natural gcosylaon of the ActRii protein so as to d iinsh the likelihood of an unfiavorabi immune response in a patient. Human and CHO cell lines have been used successfuly; and it is expected that other common mammalian expression systems wil be usef u As described herein, ActRIa proteins designated ActRlaFe (a form with a 20 minimal linker between tIe AtRsla portion and the Fc portion) have desirable properties, including selective binding to actiVn versus GDF8 and/or GDPI high affinity ligand bindi-n and serumn half life greateran two weeks in anirmalimodels In certain embodiments the invention provides ActRaic polypeptides and pharmaceutical preparations comprising such polypeptides and a phannaceutically 25 acceptable excipient. In certain aspect, the disclosure provides nucleic acids encoding a soluble activin~-indirng ActRa polypeptide. An isolated polynucleotide maiy comprise a coding sequence for a soluble, acti vim binding ActR~fa polypeptide. such as described above. For example, an isolated nucleic acid may include a sequience 30 coding for an extracellular domain (e g, ligand-binding domain) of an ActRHia and a sequence that would code lor part or all of the transne mbrane domain and/or the cytoplasmic domain of an ActRfa, but for a stop codon positioned within the -6transmembrane domain or the cytoplasmic domain, or positioned between the extraceildar domain .and the transnembrane domain or cytoplasmic domait For exanplean isolated polynuciotide may comprise a fufllengh ActRua poil'nucleotide sequence such as SEQ D NO: or 5, or a partially truncated ) verson, said isolated poiynucieotidc father comprising a transcription termnnation codlon at least six hundred nucdeotides before the 3lcminus or otherwise positioned such that translation of the polynucLeotide gives rise to an extracellular domain optionaiiy fused to a tuncated portion of a fulidength ActRIla A preferred nucleic acid sequence is SEQ 13) NO:14. Nucleic acids disclosed herein may be 10 operably linked to a promoter expression, and the Cisclosure provides cells transformed with such recombinant polynucleotides. Prefraby the cel is a manmalan cell such as a C10 cell. n certain aspects. the disclosure prove des methods fkPrnalking asouhbe, activin binding Ac~tR1 a poiypeptide Such a method may in clude expressing any of 15 the nuclic acids (ig,.SlEQ ID NO, 4 5 or 14) disclosed herein in a suitabe cel, suchas a Chin eC hamster ovary (CHO) cell. Such a method may comprise: a) culturing a cell under conditions suitable fbr expression of he soluble ActRila podypeptdev wherein said cell i tansformed with a soluble kctila expression construct; and b) recovering the soluble ActRila polypeptide so expressed Soluble 2 0 ActRila poIVpeptides may be recovered as crude, parially purified or highly purified fractions, Purification may be achieved by a seres of purification teps, including for example one, two or three or more of the following in any order: protein A chromatography, anion exchange chromnatography (eg" Q sepharose), hydrophobic interaction chromatography (e.g, phenylsepharose), size exclusion 25 chromnatogr aphy, and cation exchbangea cromatogra-phy. In certain aspects an ahtivn-ActRHa antagonist disclosed herein, such as a sohWe. activin-binding ActRUa polypeptide may be Iused in a method for promoting bone growth or increzsinfg 'one density a ut In certan embodiments, the discIosure provides methods for ircating a disorder associated 30 with low bone density, or to promote bone growth in patients in need thereof A mehod may comprise administering to a. subject in need thereof an elective amount of activin ctRfia. antagonist irn teA-tar aspects, the disclosure provides uses of - 7action. ctR.la antagonist for making a medicanent tor t' treatment of a disorder or condition as described herein, In cerisects the disosure provides a method for identifying an agent that stinuhees growth of or increased mineraization of bone. The method 5 comprises: a) identifyig a test agent that binds to activin or a lignainding domainA of an ActRua polypeptide; and b) evaluating the effec ofthe agent on growth of, or mineralization of bonae BRIEF DESCRIPTION OF THEP DRAWINGS 10 Figure 1 shows the purification of ActRila-hE expressed in C1 ele protein purifies as a singie, wedefined peak Figure 2 shows the lading of AcRa-hFc to activin and GDF- 1, as measured by BiaCore assay Figure 3 shows a schematic for the A-204 Reporter Gene Assay. The figure 15 shows the Reporter vector: pL3(CAGA)12 (described in Deier et al 1998 EMBO T7 3091-3100.) The CAGAln2 otif is present in TOF-Beta responsive ( -1ge so this vector is of genera use tor factors signaling through Smad 2 and 2. Figure 4 shows the effects of AetRla-h~c (diamonds) and ActRua-mFft 20 (squares) on ODE-8 signaling in the A-204 Reporter Gene Assay. Both proteins exhibited substantial inhibition of GD1S niediated signaling at picomolar concentrations, Figure 5 shows the effects of three different preparations of Act"imahFe on GDF- I signdainig in the A-204 Reporter Gene Assay 25 Figure 6 shows examples o.f DEXA images or control-and ActRila-mFo treated BALB/c mice, before (top panel) and after (bottom panels) the 12-week treatment period. Paler shading indicaas increased hone density Figure 7 shows a quantification of the effeas of ActRIla-mnFo on hone mineral density in BALBe mice over the 12-week period. Treatments were control - 8- (diamonds), 2 mg/kg dosing of ActRtia1nFc (squares), 6 mg/g dosing of ActRJUa mRF (Tiangles) amd 10 mg/kg dosing of ActRhainFc (circles) Figure .8 shows a quantification of the effects of ActRfa1nFc on Ine mineral content In BALB/c mice over the 12week period, Treatne we Control 5 (diamonds), 2 mag dosing of ActRtatmlc (squares), 6 mg/kg dosing of ActRha InFo (triangles) and 10 mg/kg dosing of ActRa-nF (circles) Figure 9 shows a quantification of the effects of ActRlarmFc on bone mineral density of the tra becunlar bone in ovanectomnized (OVX ) orfAhmn operated (SHAM) C57BI 6 mice over after a week period Tre atments wire central (PBS) 10 or 10 mg/kg dosing of ActRhi-Fc (ActRla), Furire 10 shows a quanttfition of the effects of Ac tRaFc on the trabecular bone in ovarectomized (OVX) C57BL6 nice over a I 2veek period, Treatments were control (PB \; pale Ias) or 10 nmg/kg dosing of Act-fia-nFC (AtRua; dark bars). 5 Figure 1.quantncatio of the effects of ActRla-ntFc on the trabecular bone in sham operated C5 IL6 mice after 6 or 12 weeks of treatmnt period. Treatments were citrol (PBS- pale bars) or 10 mggk dosing of ActRa mFc (ActRila; dark bars). Figure 12 shows the res-uits of pQCT analysis of bone density in 20 ovariectomized mice over 12 weeks of treatment Treatments were control (PBS; pale bars) or ActRIa-m~c (dark bars), y-axi: img/cemn Figure 13 depicts the results of pQCT analysis of bone density in sham operated mice over 12 weeks of treatment Treatments were control (PBS; pale bars) or ActRilam-nEc (dark bars) v--axis; mgln 25 Figures 14A aid 14B show whole body DEXA analysis after 12 weeks of treatment (A) and ex .voc analysis of fiaurs (), Light areas depict areas of high bone denity igure 15 show ex \ vivo pQCT analysis of the femoral midshaft after twelve weeks of treament freatmnents were vehicle control (PBS, dank bars) and AcR ia 30 rmfe (pale barat The four bars to the lef show total bone density while the four bars to the right showcortical bone density The first pair of bars in each set of four bars -9represent data from ovariectoinized mice while the second pair of bars represent data from sham operated mice. Figure 16 shows eX vivo pQCT analysis and diaphyseal bone contntet of the temoral rnidshafafter weeks of treatment Te e were vehicle control 5 (PBS, dark bars) or ActRUam-Fc (pale bar s), The four bars to the let show total oe content while e thur bars to the right shov cortical bone content. T'e first pair of Lars i each set of four bars represent data from ovariectomized mice while the second pair of bars represent data from sham operated mice. Figure 17 shows ex vivo pQCT analysis of the fumoral midshaft -nd feImorai 10 cortical thickness. Treatments were control (PBS, dark bars) and AcYRianmFe (pale bars) The four bars to the left show endosteal circumference while the h bars to the right show periosteal circumference, Te first pair of bars in each set of four bars represent data from ovariectomized mice while the second pair of bars represent data from sham operated mice, 15 Figure 18 depints the results of mechanical testing of femurs after twelve weeks of treatment, Treatments were control (PBSdark bars) and ActRfLmmIc (pale hars) The two bars to the left represent data from ovariectomized mice wie the last two bars represent damt from sham operated mice. Figure 19 shows the eftcts of Acithaut.lc on trabecular bone volume. 20 Figure 20 shows the effects of ctrUle 1 c on trabecular architecture in the distal femnr. Figure 21. shows the eftcs of Aorla nlo on cortical bone. Figure 22 shows the effects of AcriimFe on the mechanical strength of bone. 25 Figre 23 shows the effects of different doses of ActRIh-mFc on bone characteristics at three different dosages. Figure 24 shows bone histomorphometry indicating that ActRla-mFc has dual anabolic and anntresorptive activity - 10 - DETAILED DESCRIPTION OF THE UNVENION I. ()OT-vrvi W The tratsforming growth factor-beta (T Fbeta) sup erfamily entains a variety of growth factors that share common sequence elements and structural 5 motifi. These proteins are knowi to exr bioneeffects on a large variety of ccll type ia both vertebrate and invrtebvate Menbers of -the superfaanfiy perform mportant functions during enbryone development in pa t ten forusatio and tissue speciation and can infhiencer a vaiety of differeniation processes including adipogenesis, hnydenscsiadiogenesishematopoiesis 10 neurogenesis, and epitheial cell differentation The fantsiy is divided into vtwo general branches: the BIMP/GDF and the TGibeta/Activin!BMP10 branches; whose members have diverse, ofMn conplemenwry effect Bly mardpulating the activity of a member of tb TGF-beta family it is often possible to cause significant ph y'sological Changes in an organiFor example, the Piedmontese and Bgia 15 Blue cattle breeds carry a lss-oflfunction mutation in the GDF8(also cabled myostatin) gene that causes a. marked increase in muscle mrass. Grobet et aA Nat Genet 1997, .17(14 74. Furthermore humans, inactive alleges of GDF are associated with increased muscle mass arid, reportedly, exceptional strength, Schuelke et al, N Engl J Med 2004, 3502682-& 20 Ativins are dimeric polypeptide growth factors ma belong to the TGF-eta superfamil There are three principle activi. frms (A, B., and AB) thAt are homofheterodimers of two .osely rated 3 subunits (13 P10% a0nd [3T) The human genome also encodes an activir C and an activity E which are primarily expressed in the live In the TOT-beta superail, actv ins are unique and 25 muifumctional factors that can s timulate hornone production in ovarian and placental cos, support neuronal cell survivainfluence cell-cycle progress positively or negatively depending on ceU ype. and induce imesodernal differentiation at las t in amphibian embryos (DePaolo et alM 1991, Proc Soc Ep Big Med, 198500-512; Dyson et al 1997, Curr fiot 7,81-84 Woodruff. 1998 30 Biochemn Pharmaco55953-963) Moreover, erythroid differentiation factor (EDF) isolated from the estimated hman monocytic leukemic cWts was found to be - 11 identical to activin A (iMurata et al- 198, PNAS, 85:2434). It has been suggested that activin A acts as a naturalQ siUve regulator o erythropoiesis in the bocn mrrow. in several tissues, activin signaling is antgonzed by its related Peterodimerinhibin. For example during te release ci foliciestimulating 5 honnone (FSI!)from the pituitary. activrin promotesSH secretion and synthesis, while inhibit prevents fSH secretion ad synthesis. Other proteins that may regidate activin Ploactivity and/or bind to activin include ollistatin (PSt flistatin related protein (FR TON$ signals are mediated by heteromeric complexes of type I and type 11 10 shrine/ threonine kinase receptors which phosphorylate and activate downstream Smad proteins upon ligand stinuation (Massagu 2000. Nat, Rev. Mio. Cell Biob 1:1694178) These type I and type II receptors are trans.nembrane proteins, composed of a ligand-binding extracelhar domain with vsteinrih region a tansmembrane domhim and a oytoplasnc domain with predicted serine/hrennn 15 specificity. Type I receptors are essential for signaling; and type It receptors are required for binding ligands and for expression of type I receptors, Type I and H activin receptors form a stable complexar ligand binding sting in phosphorylation of ty pe I rceptos y type If receptors Two related type eceptors AtRfla and ActRIh have been identified as 20 the type receptors for activis (Mathews and alk, 19 9 Cell 65:973-982; Attisano etal, 1992, C01 68: 97-108) Besides aetivins Act'R ald ActRilb can biochemical interact with several Other TGF fami ionly proteins. including BMP7 NodaL DF8, and GDF1 (Yamashia et A ,1995 J Cell BioL 130:21 26; ee and Mec(herron 001, Proc Nati Acad, Sui 90:3069311 Yeo and Whiman, 25 200Mo elM 7 : 949-957; Oh et al, 2002 Genes Dev. 16:2749-54> ALk is the primary type I receptor for activ is, particularly for activin A. and ALP -,may serve as a receptor for actions as well particularly ctner B. As denonstrated herein, a soluble AtRd1a polypeptide (sActkRay which shows substantial prefierence in binding to aetlin A as opposed to other TGF-beta 30 family meberssuch as GDF8 or GDFI.is effective to promote bone growth and Increase bone density in vivo, While s to be bound to any particular - 12 mnechanisu, it is expected that the effect of sActRIa is caused primarily by an activin atagonist effect, given the very strong aetivin binding (picoolar dissociation consaparticillar sActRIIa construct used in these studies Regardless of mechanism, it is apparent fromfl the data presented herein that 5 ActRfiadativin atagonists do increase bone density i orinal mice and in mouse modes for osteoporosis. It shOild he noted that bone is a dynamic tissmeith growth or shrinkage and increased or decreased density depending on a balance of factors that produce bone and stimulate rineralization (primarily osteoblasts) and tactos that destoy and demineralize bone (prinarily osteoclasts) Bone growth and 10 mineraization bnay b increased by increasing the productie Iactors, by decreasing the desructive fact or both. The terms "promote bone growth" and "increase bone mineralization" refer to the observable physical chan ges in bone and are intended to be neutal as to the mechanism by which changes in bone occur. The niouse models fbr osteoporosis and bo.e growthdensity that were used 15 in the studies described herein are considered to be highly prictive of eficacy in humans, and therefore, this disclosure provides methods for using ActRila polypeptides and other activKnActRIa antagonists to prom ote bor gmwth and increase bone density n .ActivinActPIaY antagonists inchide, for example actiyin-binding soluble ActRita polypeptides. antbds that bind to activin 20 (particlarly the activin A or B subuits, also referred to as fIA or JIB) and disrupt ActRla binding antibodies that bind to ARfleand disrupt activin hinding.non antibody proteins selected for activin or ActRI1a hiding (see e., W/2002/O88171 WY2006/0556, V/2002/32925, WQ/2005/037989 US 200/1M3939 eand US 20/0238646 forexamples of such proteins and methods 25 for desg and seection of sarne) randomized peptides selected tbr activin or AcURIla binding, ftten afixed to an Fe domain. Two different proteins (or other moieties) with activin or Actlia binding activity, especialy activin binders that . block the type I (e g. a soluble type activin receptor) and type II (e.ga soluble type HI activin l receptor) binding sites/respectively, may be linked together to create 30 a bifunctiona biding molecule. Nucleic acid aptamer small molecues and other agents that inhibit the activin-ActRIla signaling axis, Various proteins have activity ActRIm antagonist activityincding inhibn (i-e, inhibin alpha subunit), although - 13 inhibit does not universally antrconize activin in all tissues, follistatin (e,, ftdiistacin-288 and fotli staI15), Cerberus, FSRP, endoglin, activin C, alpha(2 macroglobul and an M $A (methionine to alanine Change at position lOR) mutant actnivi A. Cenemiy alternative fbms of activin particularly those w ih 5 alterations in the type I receptor bindmig domain can bind to type 11 receptors and fail to fo an cv actave ary ompex thuIs acting as antagonists. Additionally, ir celo acids such as antisense molecules, sRNAs or ribozynmes that nhibit activin A. B, C or E or, parieuaryMUM expression, can be used as activinActR a antagonists. Preferably. the activin-ActRla antagonist to be used will exhibit 10 selectivity for ihitini activinadiated signaling versus othe mensbers if the TGP-beta iny, and particularly wvith respect to GDF8 and jDF11 Soiuble ActRib proteins do bind to activin. however, the wd type protein does not exhibit significant selectivity ht binding to activin versus GDFWI I and preihninary experiments suggest that this protein does not provide the desired effects on bone, 15 while also caus'ig substantial mas' rxovah Rowevealtered r of ActRub with diffirerut binding properties havc been idenified (see, eg, WO 2006/ 12627 pp.5559 incorporated herein by reference} and these proteins may achieve the desired effects on bone. Native or altered ActRub may be given added specificity for activin by coupling with a second activin-selective bindng agent 20 The terms used in this specification generally have their ordinary nieanings in the art, within the contexof this invention and in the specific context where each terIn is used. Certain terms are discussed below or elsewhere ignite specification, to provide additional guidance to to he practitioner in describing the cornpostOiwis and methods of the invention and how to make and use therm, The 25 scope or meaning of anyuse of a tern will be apparent from the specific context in which the term is used. Abouf" and "approximately shall generally mean an acceptable degree of error for ahe quantity eared given. the nature or precision ofthe measurements T1'ypicaty exemplary degrees of error are within 20 percent (%) preferably within 30 10% and more preferably within 5% of a given value or range of values. - 14 - Alt~enatively, and pariotdarly in biolo'il $y-tems, the t oms "about and "approxuate uma mean values that are within an order of magnitude prferably within 5-fold and more preferable within 2-fold of a given value. Ntmerical quantities given herein are apoximate unless stated otherwise, meaning that the 5 term "about" or approxhnately" can be lnfrred when not expressly stated, The methods of the in ventior nay include steps of comparing sequences to each other, including wild-type sequence to one or more mutants sequence varIants) Such comparison typically comprise alignments of polymer sequences, eg. using sequence programs and/r agorithms that ae well known in i0 the artOlt example BLAST, LSTA indMBA 6 to naunf, a few>. The skilled artisan can readuiy appreciate that in such aIgnments, where a mutation -ntas resiu insertion or deletion the sequence alignment will introduce a "gap" (typically represented by a dash, or "A") in the polymer sequence not containing the inserted or deleted residue 15 "Homologousf in all its grammatical forms and spelling vacations, refers to the relationship between two proteins that possess a "common evolutionary origin;" including proteins from superfailiies in the same species of organisms -weilas homologous Proteins from different species of organism Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence 20 similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. The term "sequence simiarir in all its grammlatcal forms, refers to the degree of identity c orepdn between nuei acid or am1ino acid sequences that may or may not share a Comnan evolutionary origin, 25 However, in common usage and in the instant application. the term "homologous, when modified with an adverb such. a "highly may refer to sequence similarity and may or may not relate to a common evolutionary origin, - 15 - 2. Acti 1a Polvopeotides In certain aspects the present invention relates to ActRfia polypeptides. As used heren, the term :AcR a" refers to a family of activin receptor type. Ha (AcRa) proteins from any species and varilants derived from such ActRik proteins 5 by mutagenesis or other modification. Reference to ActRIla herein is understood to be a reference to any one of the currently ideitfied forms Members of the ActRifa family are gcenrally trasmenbre proteins, composed of a ligandubinding extraceldiar domain with a cystcineerich region, a transmenbrane domain, and a cytoplasnic domain ith predicted serne/threonie kinase activity 10 The term "ActR1ta polypeptide includes polypeptides comprising any naturally oecurrirg poypeptide of an ActRla family member as wel as any variants thereof (inchiding mutants fragmnts, f.sins, and peptidomimetic forms) that retain a useful activity, For exampe ActRia polypeptides include poypeptides derived from the sequence of any known AcURh having a sequence at least about 15 80% identical to the sequence of an ActRia polypeptide, an ptraby at least 85%, 90%. 95%, 97%,99% or greater identity For example an Actua poiypeptide of the ivention may bind to and inhibit the function of an ActRla protein and/or activin Preebyt an A ictRla polypeptide pronotes bone growth and bone mineralizauion, Exanpes afA ctRila polypeptides include human ActRita 20 precursor polypepude (E 0if NO:) and soluble human ActRIIa polypeptides (e.g.,SEQ lD N 2: 3 7 ad 1.2) The human ActRIla precursor protein sequence is as follows: MGAAAKLAFAVFLI ISCSSGAILGRSTQLFFNAWEDRTN QTGVEPCYGDKDKtRPJCFATWKMI SGS IE IVIQGCWLDDIXNCY 25 DRTDCVSKSDS PEVYFCCCEGNMCNKFSYFPEMEVTQPTSNP VPPPtN ILLYSLtVptMLIGt IAttv~ FWVYRH:MA''YmPV cPQOGPPPSPfLOhiPLQL LEViKARtGR FGCVW;'K~ VAVKTP$IQDOSWGNEVY 'SLPGMKE'K 30 YLUZTDI PGLKDG~HKFA fSNSKvL~LK NELTAO:A05FGLA LKFEAGKSAoTGQv 1RYN AA N FT ROFL QDM YAMGLVi5WiBLASROCTAADGPVDEMLFEEGQPSEDQ V SVHKKR PVLRD WQKHAGMAMLCST OVGERTiQMQRLTNNTEDNtThVT!TNVD ;PPKESSL 35 (SEQ 1D) NO: If - 16 - The signal peptide is single underlined; the ctraellula domain is in bold and the potential Ninked glycosylation sites arc double underimed The human ActRilla soluble (extracelar ),processed poypeptide sequence 5 is as tollows: ILGRSETQECLFFN ANWEKDRTNQTGVEP GCYGDKDRRHCFAT WKN SGS IEVKQGOWLDDNtD D KDSE FC G NMCE YFSYPEMEk!TQPTSN~ PVT~PKtP r (SEQ ID NO: 2) 10 The -temnnai "tail" of the cx. acehular dolm t-n is underlined, Thie sequence with the"taiW deleed (a AlJ sequence is as folows: ILGRSETQECL FFNANEKDETN

T

QTGVE PCYGDGKDKRR C FAT WKNISGSIEIVKQGCWLDINCYDRT DCVEKDS PEVYFCCCE GNMNEKFSYFPEM (SEQ ID NO) 1$) The nucleic acid scqumec human ArM precursor protein is as hlows(nucleotidces 164470$ f Genbank ensTr\N 001616): ATGG95WGCUeGCA.A~drrocGomredd'c~n'ccTnTAcTrrczr GATGGAGA A AAACAGC W<CfTr'GAATATATTATCCT idiAGGCA~eG~d TanAGT CTOTAYWETCAuT GGAAGi~ 25 Kc Ye coTGoTr st '.'G,'.) GTxst.TGG~ TGTCAdATCACAAAGCCTACCTCCTGACTTGT CAACCAAGccCGGAC~cccCACTTCCCTTCAGTCTGAc TCTTAC2'TACGAA AnGTGG2TCAAATATcAAcrhaGAcA - 17 - TGAGGaCAG OGAGGTOOGACGTGT~CGG TAOAGrCTOAGGGTATAGGGOTCTA AAACTTCCAAGGGikAT TT ANT T AGAT G .. G T.CATC AAG GnACATTGG~cCCAX1GC ACTGAATGC AGGAAAGTTGC jQ s0x~ pN At t Idkc iA 20c c A nc 'AT (SEQ ID NO: 4) 1rhe smiliC acid sequence tenoding a human ActRi . soble (extra~elldh polypepildce is s s ollows: OOr 1 ct ItoS ter d"solubldhrte poyterm asoibed cteiti~~ nclides rl prtein T T tm"te Alrct0NAitGAGpTGTpepfd TTas se hreiATncTGGGa 25 nnlurahv'ocuriTng~etcla domamini of an actRfla prote4in as well as anIry %rr~ans tere (i P.dinn-itensfra-ments and peptidondietic forms). An act~vitindin~dk h uhvepide is one that reains -the abilitv to bind to acivin., plf AAAAAG yACAAivin AAAEAA or lT. PretrabTyGTTA ngCAt poivpentse 'WI ind tooatiin AA withagiiscito xnns-lan of It nM or'kts 30 Amin--o aceid-sequences of hanAc~m~cusrpoeni r~icUiw h extr eihdar domai dfan A'Ctdi.rtein hinds to activini and -is geerily a Anblfe. ant thus3 zr i be, termed a soh tule. atnbdigAtlaPolypepie.Exnlao soluble, asevic Actoit polypepvides inerdes the soluble poypeptide illuzrazdu in SEQ x) NCKa d2,3m 7, 12 and 13. SEQ D NOti is relferrd to as - 18 - AetRiia hFcand is described further in the ExampleS Other examples of soluble, activin-binding ActRla polypeptids compnis, a signal sequence n addition to te extraceihdar dIomin of an Authl p rotein, for exanmple. the honey'v b\e millitn leader sequence (SEQ ID NO: ) the) tissue piainogen activator (TPA) leader 5 (SEQ ID NO 9) or the native ActRfa leader (SEQ ID NO 10) The AcFIlUa-hFC polypeptide ihstrated in SEQ ID NO:13 uses a TPA leader, Functonally ctive fragments of ActUlia polypebpies can be obtained by screening polypeptides recombinantly produced fron the gorrspondin at of the nueidc aci encoding an ActRia pol ypeptlde, In additon, Fagments an be IC chemicaly sythnesized using technique known in the art such as conventonal Merrifield soid phaseMoe or t Boo chemisty. The fragments can he produced (ren y by chemical synthe'sis) and tested to identify those peptidyl fragments that can function as anagonsts (inhibitors) of ActIRa protein or signal mediated by acting 1i E unct oially active variants of ActRIa polypeptides can be obtained by screenig libraries of modified polypeptides reconbinantly produced front the corresponding mutagenized nucleic acids encoding an ActRila polypeptide The variants can be produced and tested to iden'if\- those that can funCtinsanmtagonists (inhibitors) of ActRIla protein or signaling medIated by activin. [n certain 20 embodiments, a functional vai "ant of the Ac~tRMa polypeptidescom.prises a amino acid sequence that is at Ieast 75% identieaI to an anino acid sequence selected fron SEQ ID NOs: 2 or 3. a c-er a iase sthe functialat vai-ant has an amino acid sequence at least 80% 5% 90 95%, 97%, 98t' 99% or 100% identical to an amino acId sequence selected from SEQ ID NOs2 or 3, 25 Functonal variants may he eneraed hy 'modlfying the structure of an ActRLa polypep tde fOr such purposes as enhancing therapeutic *fec or. iy (eg, ex vvo shel Ri and resistance to proteoiyc degradation in vvo) Suc h modified ActR a polypeptides when ieted to t i avin binding, are considered funtional equivalents of the naturally-ocmaring ActRila polypeptides. 30 Modified ActRla polypeptides can also be p. reduced for instance, y aino acid substitution detion1 or addition. Fo instance, it is reasonable to expect that an - 19 isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glhamatezaa th reonine with a erie or a sinWhieplacemet of an aminO acid with a structurally related amino acid ( a conservative mutaon)l willnOt have a major ef~hct: on the biogical active of theiesultin moiecide. Conservative 5 aemcmnts a those that take place Within a family of amino acids that are related in their side has, Whether a change in the amio acid sequence of an ActRlia polypepide results io a mitonal homoho cmab readily determinedb assessing the ability of the var Aci Is polypeptidc t produce a response in cells in a fashion similar to the wild-type AkRKU a poly, kpeptide. 10 T certain embodiments, the present invntion contemplates specific mutations of the ActRIe polypeptides so a to alter the glycosylation of the polypeptide Such Mutations may be selected so as to introduce or eininate one or more sis such as Oinked or Ninked glycosylition sites. -Asparaginel haked glvcosviaiomn re6'ito sites gen-eraily com,,prise a rpetd 15 sequence, asaragine-threone (or asparagines ernae) (where X" is any ainino acid) which is specifically recognized by appropriate cellular glycosyladon enzymes The aleainm~aSo be mae lty theo addition cf or s sato w one or more series or threoncinc rcidues to the sequecie of the wild4ype ActRa polypeptioe (for O linked glcosylation sites), A variety of amino acid substitutions 20 or deletions at one or both of the first or third amino acid posidons of a glycosylaton recognition cite (and/or amino acid deletion the second position) results in non-glycosylation at the modified tripeptide sequence. Another nes of increasing the number of carbohydrate moietPes on an ActRLa polypeptide is by chemical or enzymatic ouilig of glycosides to the ActRla polypeptide. 2:5 Dependig on the couplirg ndused, the sugars) may be attached to (a) arginine and histidize(b) fice carboxyl groups; (c) free sulthydryl groups such as those of cysteine; (d) frce hydroxyl groups such as those of serine, threonine or hydroxvprcVne;(e) aroma.ic residues such a' those of phenylalanineCyrosin, or trptop ;or f t group of glutamine Thes methods ared 30 WO 87/05330 published Sep 11 197 arnd in Aplin and Writn (1981) CRC Crt Rev, Biochen, pp. 259306, incorporated by reference herein, Removal of e or more carbohydrate moieties present on an ActRfa polypeptide may be - 20 accomplished chemaically andoOr enzvmatically CIeical deglycosylation may involve for e xampe exposure of the AcRUla polypeptid to the compound trifluoroethaneulfnic acid or an equivalent compoui This treatmeflit results in the 'ieavage of tst or all sugars except the iffining sugar (acetylgucosamine or 5 Nacetylgalactosamine) wle leaving the amino acid sequence intactL Chemical deglycosylation is further described by Haklmuddha e al (1987) Arch Biocben. Biophys 259:52 and Ldge et A. (198 1) Anal Biochm. 118 : E nzymnatc cleavage of carbohydrate nmoieties on ActRIa polypeptdes can be achieved byhe use ot a variey of endo and exo-glycosidases as described by Thoicura et a 10 (1987) M1th E3nzymoL 38350, The sequence of an AVct l polypeptide may be adjusted as appropdatc dependig on tb yp' e of expression used, as namnaian yeast ismct and plant cells nay all introduce differing giycosylation ar at can be affcted by the amino acid sequelne of the pep.tid n In general. ActlIla proteins for use in hmans wild be expressed in a ma Calian elline that 15 provides proper glvcosylaion such as HEK293 or C cell ilnes. although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzyrnes and insect cells are expected to he useful as well This disclosure further contemplates a method of generating mutants, particulady sets of cambinatorial mwtants of an AetRla polypeptide, as well as 20 truncation mutants; pools of combinaoriai mutants are eopecialy useful for identifying functional variant sequence. The purpose of screening such combinatorial libraries may he to generate.for example, ActRa polypeptide variants which can act as either agonists or antagonist, or alternatively, which possess novel activities all together A variety of screening assays are provided 25 below and such assays may be used to evaluate variants, For example, an ActRia polypeptide variant may be screened or ability to hind to an ActRia ligand o prevent binding of an AetRIa i gand to an AcRlia polypeptide or to interfere with signaling caused by an ActRta ligand, The activity of an Ac t RfIla polypeptide or its variants nay also be tested in a 30 cell-based or in vivo assay: For example, the effect of an ActRita polypeptide vacant on the expression of genes involved in bone prodnetion or bone destruction may be assessed. This may, as needed, be performed in the: presence of one or more - 21 recombinant ActRla ligand proteins (e g activni and cells may be transfected so as to produce an ActRlia polypeptide and/or variants mteeof and optionally an ActRIa figand tkewise, anActRa polypepide may oe adnunistered to a mouse or other an imal and one. or more hone properties such as density or volume may be 5 asset ssed. 11e lealin rate& or bone x Sfamre my also hr m\alut dDa~uegx ray absorptionetry (DEXA) is a weiestablished, noninvaswive, quantitative technique for assessing bone density in an anima, In hnamns central DEXA systems may be used to evacuate bone density in the spine and pelvis, These are the best predictors of overall bone density Peripheral DEXA systems may be used to 10 evaluate bone density in peripheral bones, including. for exampk, the bones ofthe hand wrist ankle and toot. Traditional ray imaging systems, including CAT scans may be used to evaluate bone growth and fracture healing. The mechanical strength tAbone maty also 'e evaluated Conmbnatoriall-derived variants can be generated which have a selective or 15 generally increased potency relative to a naturally occurig ActR polypepude. Likewise mutagenesis can give rise to variants wich have intaceliular ta lives dramatical ddffrent thanthe coespo.ndrig a wil type Act.RLIa poly petide For example the altered protein can he rendered either more stable or less stable to proteoMIc degradation or other cellar processes which resl in des tructon opf. or 20 otherwise inaction of a native AcRIar pol-ypeptide. Such Waiants and the genes which encode them, c.n be utrzed to aler Aetkda 3olypeptide I eves by mod.ulating thie half life of tih At tRIa polypeptides For instance a short hahife can give rise t ore transient n ca allow tighter control of recombinant ActRfa iYpoypeptie .eOves Owin the patient In an Fe fusion protein, 25 mu tations may be made in the lier (y any and'or the IP portion to i aer the haf 1ife ofthe protein A combine trial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least abortion of potential AtPilaspolypeptide sequences. For instAce, a mixture of synthetic 30 oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ActR a polypeptide nucleotide sequences are expressibie - 22 as individual polypeptidts, or attematively, as a set of larger fusion proteins (ong for p4age display) There are many ways by which the hlbrary of potatil homologs cam bQe generated fkom a degenerate oigonclectde sequeno Cemica aytesis ofa 5 degenerate gone sequence can be carried out in an automatic DNA synthesier, and the syt&ode getes then be ligated into anapprorite vector for expreson. The synihes of degenerate oliganuclertides is wel known in the at (see fo example, Narang SA (1983) Tetrahedron 39:3; Itakura et aLi (1981) Recombinant DNA, Proc. 3rd Cloveland SymposMacomolecuilesed AG Walton Amsterdan: 10 Elsevier pp7t289; Iakura et a(14) Armn Rectiochem $333;aka t al,(1984) Science 198106 k t Ial (1983) NucleicAid R i477) Such techniques have been employed in the directed evolutiolf othep r~oteins (see, for example, ScOtt et al (1990) Science 219:86390; Roberts et aL (992) PNAS USA 89:2429-2433; Devlin ot ai- (1990) Science 249: 404-406; Cwirla metal, 15 (1990) PNAS USA 87: 6378-6382; as vell as U1S. Patent Nos: 5,223 409, 5983 46 and 5 096,15), Aernfatively, otherfn of mrutagenesis cane uti'rzd Ito genemte a coibinatorial ibrary, For example AtRtiu pzo pepiei varianis can bo gener'ated and isolated tn a library y screen usi for example aan=ine scanning 20 mutagenesis and the like (Rufet at, (1994) Biochemistry 3 3:365 572; Wang et al (1994) , BioL Chem, 269:3095-3099; Balint aL, (1993) ne 137109- 118; Grodbergeta (1993) Bur L Biochem, 218:597-601; Nagashimnaet aL, (1993) 1. Biol Chem. 268:2888-2892; Lomian et At, (991.) Bice istry 301083240838; and Cunninghamn et al, (1989) Sciene 244 1081-1085), y linker scanning 25 mtagenesis (Gustin et a (1C 193)> \irlogy 193:,653a660; Brown et al, (1992) MoL Cell Bi 1 2 :2644-2652; Mknight e al, (1982) Science 232:316); by satumation mutagenesis (Meyers oft (1986) Sciene 232:61); by PC I rutagenesis (Leung etaL, (1989) Mthod Cell Mol Biol 9 or byi rndom mutaeesis, including chemical rnntagen~esis c, (Miller et at., 1992) A Short Course in Bacterial 30 Getuics, CSHL ress, Cold Spring Harbor, NY; and Greener et at., (1994) Stategies in Mol Biol 732-34), Linker scanning mnagenesis, particularly in a - 23 combinatorial setting is an attractivmethod for identWying tncated bioactivee) orrns of ActRila polypeptides. A wide range of techniques are known in the art for seeing gene products of combinatorial libraries made by point mutations and anmcations.and, for that matter, for screening cDUNA libraries for gene products having a certain property Such techniques Wi. be generaDy adaptable for rapid screening of the gene libranes generated by the combinatonai mtagenesis of ActRia polypeptides he most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable depression vectors/rnFsonnag appropriate 10 cels wvith the resulinglibrary of vectors, and expressing the combinatorial genes under conditions in wiah detection of a desired activity acilitatesrelatively easy isolaion of the vector encoding the gene whose product was detected Preferred assas include ccin bindingassays and acinedlated cen zignasay In certain embodiments, the ActRfi polypepeides fthe invention may 15 further comprise post-ranslational modhfications n addition to any that are natural present in the AtRila polypeptides Suc riodifications iude, but are not limited to, acetfao, coo arboxylaon, an phospshryiationlpation, and acylation As a resu the modifed ActRla poypeptides may contin non o acid enmrntsNuch as polyethylene glycls lipids, poly- or mn-saccharide and 20 phosphates. Eeteo ouch nonmammo aclu elements n the funeimay of a ActR&a poypeptide may be tested as described herein fi other AcTRIla polypeptide variant, \When an ActRfla polypeptide is produced in cells by leaving a: nascent ftrm ofthe ActRFia polypeptide, post transaonal processing nay also be important toconee filing and/or function of the protein. Different cells (suo as 25 CH, HLha MIDCK, 93: V1 IDANIH34T3 or HEK293) have specific cellular machine d haracterstic" mechauisms for such postdransladonal activtes and may be chosen to ensure the correct modificaton and processing of the ActRa polypeptides. In certain aspects, functional vriantso r modified forms of the ActRila 30 polypeptides include fusion proteins having at least a portion of the ActRla polypeptides and one or m.ore fusion domains Well known examples of such fusion - 24 domains include ut are not limited to, Giu~Gh. g one S transferase (OST), thioredoxin, protein A, protein G, an hum nolobulin heavy chain constant region Fc)naltose binding protein (MBP), or hmana serum albumnh A fusion domain may be selected so as to confer a desired property For 5 example. some fusion domains are particularly useful for isolation oftthe fusion proteins by afinity chromatography. For the purpose of afinity purification. relevant matrices for affnity chrorratographysuch as giutathre z mylase. and nickel- or cobak- conjugated resins are used, Many of such matrices are available in "kit" fn such as the Pharmacia G-ST purification system and the QiAe press 10 system (Qiagen) useful with (iS fusion partners As another example, a fusion domain ba he selected so as to facilitate detection of tie ActRiTa polypeptides, Examples of such detection domains include the various luorescent proteins (eig GUP) as well as "epitope tags," which are usually sho peptide sequences for which a specific antibody is available, Well kno wn eiop tags for which specific 1$ monoclonal antibodaes are readily available include FA influenza virus haemaggluinin (HA). and c myc t .I some cases the fusion domns have a protease cleavage se, such as for Factor a or Tfhroubin which allows the relevant lrotease to pardaly digest the fvion proteins and thereby liberate the recombinant oroteins rhereron. The liberated proteins can then be isolated frm the fusion 20 domain by subsequentchromatographic separation Incerta in preferred enbodiments; an ActRila polypeptide is fused with a donain that stabilizes the ActRTa polypeptide in vivo (a "stabilizer" domain) B stabilin is meoant anything that hieases serum half ifStegardles of whether tIs is because of decreasd denuuions decreased cei.arance by the kdneo or other phannacokinetic 25 effect Fusions with the po poon of an immunoglbuin are known to confer desirable phannacokinetic properties on a wide range of proteins, Likewise, usicns to human serum albumin can conr desirable properties. Other types of fusion domains that may be selected ichuIe muinierizing (et, dmeizing tetramerizing) domains and f1incatonal domains that' confer an additional biological function, such 30 as further stimlation of bone growth or muscle growth, as desired). - 25 - As a specific example the present invnition provides a fusion protein comprising a sobl extameluar domain of ActRiLa fused to an Fc domain 'e.g., SEQ ID NO, 6). THTOP C PAPE LLOG PSVFLFPPKOTLM 3SRTPEVT V A)SINDPN1I4 5 g tprue/:vN.Ygr PaQ Si VS NIQiNrK\.KCnt' VSNKAI) PvPTE TLSKAKGQPRE PQVYT I;PPSRENTKNQVSLTINKGFPPS AVEWESN GQPENNYKT T PLPVLDSDGPFF3SKLTVDKSRWQQGNVFSo S4M EAL14 A) 1'YT

QKSLSPGK

4 Optional, the Pc domain has one or more niutatlons at residues such as 10 Asp265, lysine 322. and Astu434 In certain cases, the mutant Fc domain having one or more ofthese motions (e g.. Asp- 65 mutaion) has reduced ability of binding to the FEy receptor relative to a wildtype Pc domain. In other cases. Ite mutant Fe domain having g one or more of these mutations (cg, Asam434 mutation) has Increased ability of bdngt e MIC cassrelated F creceptor (FeRN) 1 relative to a wiidtype Fe domain, t is understood that different elements of the fison proteins maybe arranged in any manner tat is consstentwith the desired t'nFtionaity. or example, an ActRila polypeptid& may be placed C-terminal to a heterologous domain, or altrnaivey a htierokogois do nain may be placed. C-terminal to an 20 AcdRia pilygpide; The ActRib polypeptide comain and the heterologous domain need not be adeiont a fWstonprotein, and additionaldoinains or ammo acid sequences my be included C-orNtermin to either domain or between the domains. in ertain embod-imlents, the AcIl polypeptides of' Te present inVefliofl 25 contain one or more modfcatons that are capable of sablizing the A ctRlla polypeptides For example, such modifications erhance the in vitro half life of the ActRil polypeptides, ebance circulatory half le ot the' AcsI polypeptdes or reduce proteolyic degradation of the ActRila polypeptfdes Schabizing modificatons inclu&, but are not limited to, fsion proteins includingn, for example, 30 fusion proteins omprising an ActRla polypeptide and a stabilizer domain, modifications of a glycosyation site (icluding, for example, addition of a - 26 glycosylation site to an ActRa polypeptide? and modifications of carbohydrate moiety (including -for example removalof carbohydrate mojeties from an ActRi polypeptide) In the case of fsion proteins, an, ActRIla polypeptide is fused to a stabilier domain such as an IgG molecule (eg an Pc domain). As used herein, he 5 term stabilizer domain" not only refers to a fusion donai (e Fc) as in the ease of fusion proteins, bt also includes nonprotenaceous modifications such as a carbohydrate noiety, or nonprotenaceous polymer; such as polyethylelycol in certain enbodiments "he present invention makes available isolated and/or purified forms of the ActRia polypeptides, which are isolated front, or 10 otherwise substantially free of other proteins. ActilRa polypeptides will generally be produced by expression from recombinant ucleic acids 3. Nucleic AcidsndiA tg a batp In certain aspects, the invention provides isolated and/or recombinat nucleic 15 acids encoding any of the ActRIua polypepides (eg soluble Ac'tRia polypepidest including fragments functional variants and fusion proteins disclosed hereim For example, SEQ ID NO, 4 encodes the naturally occurin'dhu ActRIlaprecusor polypeptide, while SEQ ID NO4' 5 encodes theprocessed extracellar domain of Ac tRla. The subject nucleic acids may be singe-stranded or double stranded, Such 20 nuder' acids may be DNA or RNA molecules lhbse nuclee acids may be used fr ". mpe n f(t o~i r'lmaking AetItRRa polvpentides , or as direct therpei& agxn t (egin a gene therapy approach) in certain aspects the> sjec nucleic atias encoding ActRita p.olypeptides are farther understood to nlAde nucleic acids that are variaits of SEQ D NO 4 or 25 5 Variant nucLeotide sequences inc ude sequences that diffYer by one or more nucleotide substitutions, additons or deletions, such as altebi variants, in certain embod aiments. the invendion provides isolated or recainbinant nucleic acd sequences that are at least 80%. 85, 9095%, 9 % 98%, 99% or 100% identical to SE Q ID NO: 4 or 5 One of fordina still in th art will 30 appreciate that nucleic acid sequences complementary to SEQ ID ND: 4 or 5, and varianms of'SEQ ID NO: 4 or 5 are also within the scope of this invention in further - 27 einbodiments, the nucleic acId sequences of the invention can be isolated, recombinanw and/or fused ith a heterohogous nucleotide sequence, or in a DNA Hibrarv. in other embodiments, nucleic acids of the invention also include nucleonde 5 sequences that hybridize under higlly stringent conditions to th e nucleotide sequence designated in SEQ ID NO: 4 or, comp enen sequence of SBQ ID NOt 4 or , or frWmnts thereof As discussed above, one of ordinary skill in the art will understand ready that appropriate stringency conditions which promote DNA hybT ideation can be Vri On of ordinary sil in the it wl understand readily t0 that appropriate stringency condtons which promote DNA hybridizatioa can be vared For example.one could perform the ridizatlion at 6J) x sodium chloride/sodium citrate (SSC) at about 45 C, fllowed by a wash 2f0 x SSC at 50 "C, For example, the salt eoncentrton in the wash step can be seleted from a low stringency of about 2.0 x SSC at 50 C to a high stringency of about 0,2 x SSC at 0 15 "C in addition, the te.mperature in the wash step can be increased ftom low conditions at room temperature about 22 OCo Ig stringen y conditions at abou 65" Both temperature and salt may he varied or temperature or salt concentration may be hldc constant while the other ab is changed. In one enodiment ther invention provides nucleic acids which hybridize under low 20 stringency andiions of 6 x SSC at room temperature Illowed by a wash at 2 x SSC at room temperature: Isolated nuleic acids which differ from the nmleic acids as set forth in SEQ ID Nts 4 or 5 due to degeneracy in the genetic code are also within the scope of the invention For example a manb er of anino acids are designated by nore than ae: 25 triplet .odo that spec fy the same amino acid, or synonyms (for example. CAU and CAC are synonyms for histidine) may result in silent" mutations which do not affect the amino acid seoence of the protein. However . i s expected that DA sequence polymorphisms that do lead to changes i the amino acid sequences of the subject proteas Wilt exist among namnalian celLs One skilled in the art wil 30 appreciate that these variations in one or more nucleottdes (up to about 53% of the nuoleotides) of the nucleic acid encoding a particular protein may exist anong - 28 individuals of a given species due to natmal alelic variation, Any and all such nucleotide variations and resulting amino acid polymiorphisns are wMithin. the scope of this invention. In certain embodiments, the recombinant nuceic acids of the invention may 5 be operably linked to one or more regulatory nucleotide sequences i an expression construct Regulatory nucleotide sequences Wi generally be appropriate to the host cell used for expression. unerous types of appropriate cexpression actors and suitable regllatory sequences are known in the art for a varicy of host els, Typical lly, said one or more regulatory nucleotide sequences nay include, but are 10 not limited to, promoter sequences, leader or signal sequences, ribosonmal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible protemrs as known in the art are cotemplated by the invention. The promoters ma be either naryoccurring promoters or hybrid promoters that 15 combine ofeens ofiore than one promoter. An expression construct may be present in a cell on an episoneN s-uch. as a plasmid, or the expression construct may be inserted in a chronosome. in a preferred embodinent the expression vec t or contains a selectable marker gene to allow the selection of transfbrned host cells, Selectable.nafker genes are well known in the art and will vary with the ho)st celi 20 used, in certain aspects of the invention the subject nucleic acid is provide in an expression vector comprising a nucleotide sequence encoding an AcfRla polypepfide and operably Hnked to a east one regulatory sequences Regulatory sequences are arwarecognized and are selected to direct expression of the Actila 25 polypeptide: Accordingly the term regulatory sequene includes promoters enhancers. and other expression control elements, Exemplary regulatory sequences are described in Goctddt ;ene Lxpresm Tchnology: Artdh inymologv Academic Press, San DiegoOA (1.990) For instance. any of a wide variety of expression control sequences that control the expression of a DNA sequence vhen 30 operatively linked to it may be used in these vectors to express DNA sequences encoding an ActRila polypeptide. Such useful expression control sequences, include, for example, the early and late promoters o SV40, tet promoteradenoirus - 29 or cytcmegalovirus inmediate earl promoter, RSV promoters, the lao system, the trp systen, the TAC or TRC systerTi, promoter whose expresson 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 ~phosphoglycerate kinase or 5 other glycolytic enzymes the promoters of aId phosphatase, eg PhoS, the promoters of the yeast anM ting factors the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic colls or their viruses, aid various combinations thereof It should be understood that the design of the expression vector may depend on such factors as 10 the choice of the host cell to be transformed and/or the type of protein desired to be expressed' Moreover the vectors copy nmer, the ability to control that copy number and the expression of any otherprotein encoded bythe vector uch as antibiotic markers, should also be considered. A recombinant nucleic acid of the invention can be produced by plating the 15 doned gei, or a portion thereof nt a vector suitable for expression in either prokaryotic cells ukaryotc cells (yeast, avian. insect or mamalian\or oth Expression vehIcles for production of a recombinant Actiia polypeptide include plasmnids and other vectors. Por nstace.suitable vectors include plasmids of the types: pBR-22-derived plasrids pEMBL-derived piasnid, pEX-derived pksbnds, 20 pBTac-deri'ved plasmids and plCderived plasmids for expression in. prokaryotic cels such as A cOli. Some rmammain express sion vectors contain both prokaryofic sequencesto fIte the pnatfon of th ei ctr in bac ter ia, and'm on r mnore eukarvotie tratisctrption units that are expresed in eukaryotic cel s The pcDN Ala'mp, 25 peDNA'nec 'P"R/CMV pSV'gpt, pSV2neo. pSV2 dhro nkSneo, pKMSOt pSVT, pkonneo and pHyg derived vectors are esanples of manma expression vectors suitable for trausfetion of eukaryotic cells. Some of thse v are modified wth sequences from bact erial plasmids, such as pBR322 t facil.atc repication and drug resstance selection in both prokaryotic and eukaryo t c" 30 Atern'atively, derivatives of viruses such as the bovine papilloma virus (BPV4), or Epstein-Barr virus (pHE pREP-denved and p 2 0 5) can be used for transient expression of proteins in eukaryotic cells, Examples of other viral (including - 30 retroviral) expression systems can he fobud bekw in dscription of gene therapy delivery systems. The various methods employed in the preparation ofthe plasmid and in transformation of h ost organisms are well known in the art' For other suitable expression systems for both prokaryotic and eukaryotic cells, as wel as general 5 recombinant procedures. see Molecua loning A Laboratory MAdnuah 3r d, ed, by Sambrook, Fritsch and Manitis (Cold Spring Harbor Laboratory Press, 2001) In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovinus expression systems include p'V derived vectors (such as pV-i392, pVLI 393 and 10 pVt94I1), pAcUW -derived vectors (such as pActIW1) and pIliueBacderived vectors (such as the Bigai containing pBlueBac ilI). In a preferred embodiment, a vector will be designed for production of the subject ActRIla polypeptides in CO cells, such as a P mv-Script vector (StratageneLa Jo a, Calif) pcDNA vectors (Invitrogen, Carlsbad. Calif.) and 15 pCIbneo vectors (Promega, Madison, Wisc.). As wvill be apparent, the subjet gene constructs can be used to cause expression of the subject ActRila polypeptides in cells propagated in culure e'g, to produce proteins, icunfsn proteins or variant proteins, for purification. This disclosure also pertains to a host cell transfected with a recombinant 20 gene nc uding a coding sequence (eSg SEQ ID NO: 4 or 5) for one or more of the subject ActRia polypeptides. The host ceml may be any prokaryotic or eukaryote cel For example, an ActRUba polypeptide of the invention may be expressed in bacterial cells such as h co&i, insect cells (eg, using a baeulovirus expression system) yeast, or mamInalian cells. Other suitable host cells areknown to those 25 skilled in the art, Accordingly, the present invention further pertains to methods of producing the subject ActRlia polypeptides. For example, a host cell transfeted with an expression vector encoding an ActRila polypeptide can be cultured under appropriate conditions to allow expression of the ActRilipolypeptide to occur The 30 ActRila pciypeptide may be secreted and isolated fron a mixture of cells and medium containing the ActRfla polypeptide. Alternatively, the ActRIa polypeptide -31may be retained cytoplasmicahly or in a membrane fraction and the cells harvested, lysed. and the protein isolated A cll cuiture inchides host cells media Ind other byproducts Suitable inedia fIor ell iuure are well known in the art The sudject ActRda polypeptides can be isolated from cell culture medium, host cels or both, 5 using techniques known in the art for purifying proteins, including on'uxchange cbomatography gel tfihration Chromatography ultritration, electrophoresis, nnnaffiniy purification with antibodesNpez.cific for particular epiopes of the ActRJla polypeptides and affinity puiication with an gen that binds to a domain fused to the ActRha polypeptde (e a protin A. column may be ussd to puify au 10 ActRlaac fusion In a preferred einbodiment the ActRla polypeptide is a fusion protein containing a domain which facili t ates its purification, In a. preferred embodiment, purification is achieved by a series of column chromatography steps, incudrig for example, three or more of the folowing, in any order: protein A chromatography Q seplharose choniatkgraphy, phenylsepharose chromatography 15 siz exc lusion chromatography and -ation exchange clromatograpy, The purification could be completed with viral filtration and buf&r exchange. As demonstrated herein ActRIUadhFe protein was purified to a pirity ofm98% as determined by size exclusion chromatography and>95% as determined by SDS PAGE, 1 hiseve of purity was sufficient so achieve desirabc effects on bone in 20 mice and an acceptable safety profile in mice, rats w ran onhuan 4 primates. In another embodiment, a fusion gene coding for a purification leader sequence, such as a poOis)/enterokinase cleavage sie sequence at the N terminus of the desired portion of the rcombinant Actiiia polypeptide, can allow purincatlfim of the ex-,preswed fusion pro-tein by affiniy chrmatography using a lip 25 metal resin., The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the buried ActRa polyeeptide (e se Hochuli et at, (1987) £ Chromatogmpb' 411l1; and Ianknecht et at A US< 88:8972) Thfchniques for making fusion genes are well knowr. Essenrti=alhye j. isi 30 of various DN' fragments coding for different polyppt ide sequences is performed in accordance With conventional fechnques, nploying bhmt-ended or stagger ended ternini for igatio n restrietlo enzyme digestion to provide for appropriate - 32 teflt 4 Iil 1ng in Ot cohesive ends as appropriate, alkaline phosphatase treatment tco avoid undesirable joining, and enzyatif ligation. i another embodiment. the fusion gene can be synthesized by conventional techniques iNciAding automated DNA sythesizers. Alternatively, PCR amptifcauion of gene fragments can be 5 carried out using anchor primers which giv tixe t complementary overhalngs between two consecutive gene ragnents which can subsequently be annealed to generate a Chaneric gene sequence (seetfot example Current Protocols in MAlecudar Biology eds. uAsubel et al. John WUey & Sons: 1992), It) 4. Alernatve Activin andeAcRf(Lataeigsts The data presented herein demonstrates that antagonists of acti in-ActRITa signaling can be used to promote bone growth and bone ninmralzaton Althongh soluble ActRIa polypeptides, and particularly ActrilaFa, are prefered antagordsts and akthougb such antaonists may affect bon' through a "nedhanism other than 15 activt antagonisi Qga atvn inhibiion may be an indiaya of the tendency of an agent to hibi 'he actwives of'a spectrmn of molecules incuiw'ng perhaps otier menibers of the TG beta superamiiy, and such collective inhibition may lead to the desired effect on, boneother types of actwin-ActRla antagnits aire expected to be useifulincluding antiactivin (eg A 1 C or E) tibodies, anti-ActRlla 20 antibodies, antisense. RNAI or niozyme nucleic acids tt inhibit the prodtiottn cf ActRila and other inhibitors of activin oYr ActRila particuladby those that disrupt activinActRUa binding. An antibody that is seficalhy reative with an AcRll poaypetde ga soluble Adidla polypeptide) and vi'h eithe binds competitively to ligend with l the 25 ActRia polypeptide or otherwise inhihits AtRrlhtediate sinaig my be used as an antagonist of AItRI~ poyepie civtes likewise; an antibody that is specifically reactive with an activin A polypeptide and which disrupts ActRita binding t'itg may used as an antagornist, By using imnmunogens derived from an ActRfta polypeptide or an activin 30 polypeptide anti-protein/anti-peptide antisera or inonoclonal antibodies can be made by standard protocols (see, for example Antibodies: A Laboratory Manua ed, by - 33 - Harlow nod Lane (CkI Spring Harbor Press. I 988)), A mammal, suh as a mouse, a hanster or rabbit can be inununized with an imunogenic fbn of the ActRfla polypeptide, an aigenic fragment which is capable of eliciting an andtbody response, or a fusion protein. Techniques tor conterrng immunogeniity on a 5 protein or peptide include conjugation to cArriers or other techniques well known in the art, An jmnrmogerdu portion of an ActRIfa or activin polypeptide can be administered in the presence of adjuvant, The progress of imnunization c'an be monitored by detection of antibody titers in plasnm or senm Standard ELSA or other inmunoassays can be used with the imunogen as antigen to assess the levels 10 of antibodies. Following immunization of an animal w ith an antiigenic preparation of an AcRlla polypeptide, antisera can be obtained and, if desired, polyconal antibodies can be isolated from the serum. To produce monoclonal antibodies antibody pro ducing cells (ymphocytes) can be harvested from an immunized animal and i5 fused by standard somatic cel f -sion procedures wit immortalizing cells such as anyeloma cel2 to yield h.i.ydona oells- St techniques are well known in the art, and inebde, for example, the hybridonna technique (originally developed byKohier and Milstein (197) Nature, 256: 495-497), the human B cell hybddoma e iq Kozhar etal (1983) Irnnology Today.4; 72 and the EB-iybridoma 20 technique to produce humannmonoclonal antibodies (Cole et al, (1985) Monocional Antibodies and Caneer Therapy, Alan Rhiss, bIoc pp,7-96) Hvbrioma cells can be screened imanankochenmically for production of antibodies specifically reactrve with an AtIUla polypeptide and onoconal antibodies isolated from a culture comprising such hybridma cells, 25 The teru "antibody" as used herein is intended to include fragments thereof which are also specifically reacive with a subject polypeptide Antibodies can be fragmented using conventional techniques and the fragnents screened for utility in the sane meaner as described aboVwfor whole antibodies. For example. Fab) 2 fragments can be generated by treating atib dy weth pepsin, The resulting F(ab)2 30 fragment can be treated to reduce disulfide bridges to produce ab fragments. The antibody of the present invention is irther intended to include bispecific, single, - 34 chain, chinerin;. hmwanized and fully hunan moliecues having affinity for an Actla or advin polypeptide conferred by at least one CIR region of dhe antibody. An antibody may further comprise a label attaced thereto and able to be detected (e g, the label can be a radioisotope; fluorescent compound, enzyme or enzyme co 5 factor) In certain embodimentsthe antibody 1s a recominant antibodv which tera encompasses any antibody generated 1 part by techniques of molecuI'ar biology, inchiding, CDR-grafted or chlineric antibodies. numan or other antibodies assembled from libraryvselected antibody domains, single chain antibodies and single donain 10 antibodies (e., hunan V proteins or camelid Vw proteins). in certain embodiments an antibody of the invention is a nmonocinal antibody, and in certain embodiments, the invention makes available methods for gnerating novel atibosdie. , For example method for generatng a monotlonal antibody that binds especially to an ActRila polypeptide or activin polypeptide may comprise 15 administering to a mouse an, amount of an imnunogenic conlpostion comprising the antigen potypeptide effective to stinnlate a detec table innmne response, obtaining antibody-producing ces (e g, cellsfom the spien) from the mouse and fusing the antibod-producing cells with nyelama cells to obtain antibody-producing hhybridomas, and sti antiodprodcing hybridonas identify a hybridoma 20 that produces a monocokonal antibody t bInds specifically to the antigen, Once obtain d. -a hybridorna can be propagated in a cel culture, optioa in u~cuture conditions where the hybridomaderived cells produce the nonoclanal antibody that binds specifically to the antigen. The monoclonal antibody may be purified from the cell culture. 25 The adjective "specificaly reactive with" as used in reference to an antibody is intended to mean, as is genera ly understood in the art, that the antibody is sufficiently selective between a enen of interest (eg an Acua polypeptide) and other antigens that are not of interest that the antibody is useful for. at ninirnun detecting the preseieof of the oafigen t interest in particular type of biological 30 samnpl. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity iN b ing may be desirable Monoclonal antibodies generally hae a greater tendency (as compared to polyclonal antibodies) - 35 to discriminate effectively between the desired antigens and crosseatting polypeptides One characteristic that influences the specific ty of an antiody:antgen interaction is the affinity s the aibody for the antigen Although the desired specificity may be reached with a range of differentaffinitis generally preferred antibodies will have an affnmiy (a dissociation constant) of about I 10i 10t 10 o ~r less (Given the extraordinarily tight binding between activin and ActRua it is exected that a neutralizing atactin or anti-ActRlia antibody would generally ha'e a dissociation constantof I orless. In addition, the techniques used to screen antibodies in order to identify; a ,10 desirable antibody may inriuence the properties of the antibody obtaied For examples' antibody is to be used for binding an antigen in solution, it thay be desirable to test solution bidinc A variety of different techliqtues are available for testing interaction bet'eee antibodies and antigens to identify particularly desirable antibodies, Such techniques ncude ilISAs, surface plasnon resonance binding 15 assays (eg, the Biacore 't binding assay, iacore AB, ppsala, Sweden), sandwich assys (eg., the paramagnetcica bed syem of 1GEN ltternational, Inc, Gaithersburg, Maryland) western blots, immunopriation assays, and immiunohistochemsistry. Examples of categories of nucleic acid compounds that are activin or 20 ActRia antagonists include antisense nuclic acids. RNAi constructs and catalytic nucleic acid constructs. nucleic acid compound ay be single or double sanded A double stranded compound may also include regions of overhang or non complementaity where one or thc other of the strands is single stranded, A single stranded compound may include regions of selfomplementarity meaning that the 25 compound forms a so-called "hairpin or ' stenmloopv structure, with aregion o1 double helical srructure A nucleic acid compound may con. rise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than OO no more thn 250. no more than U10 or no iore than 50, 35, 30, 25, or 1.8 nucl eAid es of 3" the!''u fb l i Akcr .. Mw nokic 30 PA or activin p3b nucleic acid sequence. The region of complenmentarity will prefterably be at least 8 nucleotidesand optionally at least 10 or at least 15 and optionally between 15 and 25 nuleotides A region of - 36 cutmplmentarity may faithin a'In intron, a coding sequence or a oncoming sequence of tie target tMascript Such as the coding sequece portion Generally a aceic acid compound wi I have a length of about 8 to about 500 nuakotides or base pairs in length, and optionally the lengt will be abodt 14 to about 50 5 nucleotides, A nuclei, acid may be a DNA (particuly for use as an antisense) RNA or RNA:DNA hybrid Any one strand may include a nAxture of DNA and RNA. as veil as modified forms that cannot readily be classified as either DNA or RNA. a double stranded compound may be DNA;DNA ,DNARNA or RNA:RNA and any one strand may also include a mixture of DNA and RNA.as 10 wel as modified forms that cannot readily be classikJ as either DNA or RNA, A nueic acid compound may include any of a variety of modifications including one orod tcations toe backbone (tie sugar-phosphat pordoo in a natural nucic acid, includingintemucleotde linkages) or the base portin (the purie or pyrmkdine portion of a natural nucleic acid) An andsense nuclic acid. compound 15 will preferably have a length of about 15 to atout 30 nac tides and will often contain one or more modifications t improve characteristis such as stability in the serurm in a cell or in a place vhem the compound is likelyto be delivered. such as the stomach in the case of orally delivered compounds and the tung for inhaled compauds, ia the case ofan RNAi construct the strand complementary to the 20 target transcript ill generally be RNA or modifications thereof, The other strand nmay be RNA, DNA or any other variation, The duplex portion of double stranded or singe stranded "hairpin RNl' i construct viil preferably have a kegth of IS to 40 nuclotides in length and optionally about 21 to 23 nueleotides in length, so long as it serves as a Dicer substrate. Cataiytt or enzyrmatic nucleic acids may be 25 ribozymes or DNA enaynmes and may also contain modified forms Nuciei acid compounds may inhibit expression of die target by about 50%, 75 90% or more when contacted with cels under physiologicaconditions and at a concentration where a nonsense or sense coxtrol ha~s little or no effect. Preferred concentrations fort g st ne e t of nuclei aid pounds are 1, 5 and 10 nicromola Nucleic 30 acid compounds may also be tested for effects onfr examplebone growth and mineraliztion, - 37 - S Screenintg Assavs i certain aspect the present in vention rellates to the use of ActRla polypeptides (e ,g, soluble ActRIla poiypeptides) and activin polypeptides to identify compounds (agents) which are agonist or antagonists of the activin-ActRYa signaling pathway Compounds identified rough this ascreeaing can be tested to assess their aility to modulat bone grNowth r mili o i Optionally these com dcan father be t d in anina nIdels to a s their ability to modulate tissue growth in vivo There are numerous approaches to screenia f therapeutic agems for 10 modulating tissue growth by targeting activin and Acifa polypeptides. In certain embodiments, highdhroughput screening of compounds can be carried out to identity agents that perturb activin or .ActRila-mediated eftoets on none. In certain embodimecnts the assy Is carried out to screen and identify compounds that specifically inhbit or reduce binding of an ActRila polypeptide to activin 15 Afternatiively to assay cn be used to identify compounds that enhance binding of an AetRHla polypeptide to active. i a fthe embodiment, the compounds can be identified by their abinty to interact with an actvin or ActRla poiypeptide. A variy of assay formats will suffice and.,in eight of the present disclosure, hose not expressly described herein will nevertheless be o prehended by one of 20 ordinary skill in the art. As described herfinltthe test compounds (agents) of the invention may be created by any conbinatorial chemical method. Alternatively, the subject compounds may be naturally occurring bicmolecules synthesized in vivo or in vitro. Compounds (agents) to be tested for their abulty tou act as modulators of tissue growth can be produced, for by bacteria, yeast, pIants or other 25 organisms (eg, natural products produced chemically (V g, samiainolecudos, including peptidomirneti) or produced recmbinantly. Test compounds contemplated by the present invention include nonpveptidyl organic molecules, peptides potypeptides; peptidonmictics; sugars. huomonesi and nucleic acid nmolecules In a specific embodiument, the tst aget is a small organic molecudo 30 having a molecular weight of less than about 2,000 daltons - 38 - The test compounds of the invention can be provided as single, discrete entities, or provided in libraries of greater complexity such as made by combinatorial chemistr. These libraries can comprise, for example. acohoisayl halides.nines. aides, esters, akdehydes, ethers and other classes of organic 5 compounds, Presentaton of test compounds to the est -ytem can be in either an isolated form or as mixtures of compounds, especially in initial screening steps. Optionally, the compounds may be opt;nally derivatized with other compounds and have derivatizing groups that facilitate isolation of thecompounds; Non-liniting examples of g ups inchde biotir fluorescein, d4igxygenin green 10 fluoresce-t protein, isotopes polyhistidine. magnetic beads, aglutathione S transferase (GS) photoactivatibie crossiinkers or any combinations thereof In mIay crg Screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given peiod of time. Assays which are 15 performed in ceifree systems.such as ray be derived with purihed or semi pur ied proteins, are otten preferred as "prinar" screens in that they can be generated to permit rapid development and rel-tively easy detection afan aiheration in a molecular target which is mediatd by a test compound Moreoverthe effects of ceihtar toxicity or bioavallability of the test comporind can be gorealy ignored 20 in the in vitro system, the assay instead being focused prinarily on the effect of the drug on the molecular target as may be msaniftA in an alteration of binding afty. between an AotRita polypeptide~and activin Merely to illustrate in an exemplary screening assay of the present invention, the con-mpo'und of interest is contacted with an slated and purified 25 ActRita polyp-'eptide whih is ordinarily capable of binding to activin. To the mixture of the compound and ActRia polypeptide is then added a composition conaining an ActRita ligand. Detection and quantification of ActRia/activin complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the ActRita polypeptide and acivin. 30 The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations ofthe test cornponnd Morover, a control assay can also e performed to provide a baseline for com parison.Fo - 39 example, in a control assay, isolated and puriied activin is added to a composition containing the ActRita polypeptide, and the foraStion of ActRilaactivin complex is quantitated in the absence of the test compound, It will be understood that, in general, the order in which the reactants may vbe admixed can be varied, and can be 5 adixed simultaneously. Moreover, in place of purified proteins, cellular extracts and lysates may be used to render a suitable cell-free assay system. Complex formation between the ActRa polypeptide and activin may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (eg P S C or ); iorescentiy labeled (eg ffTTC)or enzymaticaily labeled ActRia polypeptde or activin.by immunoassa or by chromatographic detection. In certain embodiment, the present invention contenlates the use of fluorescence polarization assays and fliorescence resonance energy transfer (FRET) 15 assays in measuring, either detrcdy or indirectdy, the degree nof eraction between an ActRfla polypeptde and its binding protein. Further, other modes of detection. such as those based on optical waveguides (PCI Publication WO 96/26432 and U.S, Pat. No. 5,677,l96). surhe plasmon resonance (SPR), surface charge sensors, and surfae force sensors, are compatible with many embodiments of the invention. 20 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 potntiate interaction between n ActRa polypeptide and its binding protei. See for example, U S Pat, No, 283317; Zervos etal(1993) Cell. 72:223 -232; Madura et at (1.993) J Uiol Chem 268:12046-12054, Bartel et al. (1993) Biotechniques 25 14:920-924; and Iwabuchi et at (1993) Oncogene 8:16931696). In a specific embodiment, the present iwetion contemplates the use of reverse two hybrid systems to identify copud e .g. small molecules or peptides) that dissociate interactions between an ActRilua polypeptide and its binding protein. See for exarnple, Vidal and Legrain (1999) Nucleic Acids lRes 27:919-29; Vidal arid 30 Legrain, (1999) Trends Biotechnol 17:374-81; and SPat. Nos 5, 525490; 5,955 280; and 5 965.368, - 40 - In certain embodiments the subject compounds are identified by their ability to interact with an ActRila or activin polypeptide of the invention The interaction between the compound and the AotRIfa or activin polypeptide may be covaient r For example, such Interaction can be identified at the protein lcvel 5 using in vitro biochemical methods, including photo-crossiking, radiolabekd Hlgand binding, and affinity chromatography (Jakoby WB et al, 1974 Methods in Enzymology 461 ) In certain cases, the compounds may be screened in a mechanism based assay, such as an asay to detect conpounds which bind to an activia or ActRla polypopOde. This may inoyde! a solid phase or fluid phase 10 binding event Aromatively, the gone encoding an activio or ActRlia polypeptide can be transfected wit a reporter system (etgJ$galaosidase. luciferase or green fluorescent protein) nto a cea eed mg st the library preferably by a high througnhput screening or wn rh individual nemubers of the library. Other ineehanisnm based binding assays muy be used, for example, binding assays which detect 15 changes in free energ. finding assays can be performed with the target fixed to a well bead or chip ar capured by an imimobizied antibody or resolvedby capillary electrphoreis. T. he bound compounds may be detected usually using coloimnetrc or fluorescence or surface piasm~on resonance. In certain aspects, the presn invention provides methods aid agents for 20 modulating (stimulating or irihibiting one formation and increasing bone mass. Therefore any ompound identified can be tested in whole cells or tissuesin vitro or in vivo; to confirm their ability to modulate bone growth or mineralization, Various methods known in the art can be utilized for tis purpose, For example, the effect of the AcfRJilla or actv-in polypeptdes or test 25 c-pompouds on bone or cartilage growth can be ed y neasurng induction ofMsx2 or differntiation of osteoprogenitor cells into osteoblasts in cell based assays (sec e g Dauiski et at Nat Genet 2001, 27(l4$84~8;no et al, Front Biosci. 2004 9 2 019) Another example of celobased assays includes analyzing the osteogenie activity of the subject ActRIla or activin polypeptdes and test 30 compounds in mesenchma progenitor and osteoblastic cells. To illustrte, recombinant ienoviuses expressing a'tivin or ActRia polypeptide can be constructed to infect pluripotent meseuchyna progenitor C311 CtT/2 cells, - 41 prtosteoblastic C2C12 cells, and osteobastic TE 85 cells, Osteogenic acivity is then determined by measuin the induction of alkaline phosphatase, osteocalcin, and matrix mineraliation (see, e g Cheng et al, J bone Joint Surg Am. 2003 85~ A() 44-) S The preSent invention also contemplates in vivo atssays t measure bone or cartilage growth, For example Nanking'Matthai et al, Bone, 2880-86 (2001) discloses arat osteoporotic model in Wich bone repair during the eary period after fracture is studied, hbo dt Steroid Biochemistry & Molecular Biology 68:197 202 (1999) also discloses a at osteoporotic mode Iin which bone repair during the ( lakte period after fracture is studied. Andersson et al, I. Endocrino ,170:529537 describe a mouse osteoporosis model in which :miceae ovariectoized which causes the mice to lose substantial bone mineral content and bone mineral density, with the trabecular bone losing roughly 50% of bone mineral density, Bone density could be increased in the ovariectomized mice by administration of factors such as I5 parathyroid hormone. In certain aspects, the present utwention makes use of tracture healing assats that are known in the art These assays include fracture technique histological analyst is, anid biomnecha nical analysis. whi are described in Aer example RI Pat, No, 6,521.750,ich is incorporated by rerence in its enifrety fOr its disclosure of experimental protocols for causing as wed as measuring 20 the extent of fractures, and the repair proce ss; i Emolarv Therapeutic U.ses In certain mbimenhints, actwin-ActRita antagonists (eg.,. ActRila polypeptides) of the preseat invention can be used for treating or prevenig a 25 disease or condition that is associated with bone damage. whether egbrough breakage, loss or denera on In certain embodinents, the present invenion provides methods of ring or preventing bone darnage in an ndividua in ned thereof through administring to the individ ul a therapeutically effketive amount of an aCtin~-&- k! antagonist particularly an Actfila poypeptide in certain 30 embodinns the present invention provides methods of promoting bone growth or inieralization in an individual in need thereof through administering to the - 42 individual a therapeutically effective amount ofan activin-ActRIIa antagonist, particularly an ActRIL poiypeptide These methods are preferably aimed at therapeutic and prophylactic treatmIents of aniads ,and more preferably, humans In certain enbodiments the disclosure provides for the use of activin-Actla 5 antagonists (particularly soluble A otRIapo eptides and neutralizing antibodies targeted to ecivia or ActRla) tbr the treatme nt of disorders associated with low bone density or decreased bone strength As used herein a therapeutic that "prevents" a disorder condition refem to a compound that; in a statistical sample reduces the occurrence of the disorder or 10 condition ithe treated sample relative to an untreated control sample, or delay's the onset or red ucesdht severty of one or more symptoms f sorder or condt ion relative to the untreated control sam p 1e, 'heterm treating" as used herein inlhdes propitlaxis ftnam condition or ameloation or elimination 0f the condition once it has been established I eiter case, prevenion or treatment may be 15 discerned in the diagnosis provided by a physician and the intended result of administration of the therapeutic agt The disclosure providers methods of inducing bone and/or cartilage nation, preventing bone loss, increasing bone mineralization or preventing the demineralhzation of bone; For example, the subject activin-ActRiki antagonists have 20 application in treating osteopor 'osis ad the heading of bone fractures and cartilage defects in humns and other animas ActRia or activin polypeptides may he usefid in patients that are diagnosed with hbcBinka low bone density, as a protective measure against the development ot osteoporosis. in one specific embodiraent'methosand conpositions of the present 25 in-vention may find medical utility in the hearing of bone acres 'and cartiage defects in humans and othr anima 'The subject methods and. compositions may also have prophyla tIC use in losed as well as open fracture reduction and also in the improved flxation of artdifaoints De novo bone formation induced by ai osteogenic agenxcontribuis to the repair of congenital, trauma-induced, ar 3 oncologic rejection induced craniofaciai defects, and also is usefu Ain cosmetic plastic surgery. In certain casesthe subect activindAtRfa antagonists may - 43 provide an environment to attract bonedorming cels stimu late growth of bone forming cells or induce differentiation of progenitors of honetfrining cells. Activin-ActRI~a antagonists of te invention may also be usethi in the treatment of osteoporosis. 5 Methods and compositions of the invention can be applied to conditions characterized by or causing bone 1oss, such as osteoporosis (inchiding secondary osteoporosis), hyperparathyroidisn. Gushing's diseasePagets disease, thyrotoxicos ischronic diarrheal state or nalabsorpton, renal tubular acidosis, or anorexa nervosa, 10 Osteoporosis may be caused b, or associated with, various factors. Being pariculay a post-menopausal felet h vng a low body weight, an d leading a sedentary lifestyle are allrisk factors for osteoporosis (loss of bone niineral density. leading to fraturerisk) Pcrsons having ay of the allowing profiles may be candidates fr Vreannent with an ActRla antagon ist pst-nopausal wornaln .15 and not taking estogen or other hormone replacement tirapy; a person with a personal or natemal histor hip fracture or smoking; a post-lm-enopausal voman who is ta (over 5 feet 7 inches) or thin (less than 125 pounds) a man with clinical conditions associated with bone os s; a person using medications that are known to cause bone loss: including corticosteroids such as Prednisone

T

various antisizure 20 medications such as Dilantin and certain barbiturates, or high-'dose thyroid replacement dngs; a person having type 1 diabetes liver disease. kidney disease or a flanaily history of osteoporosis; a person having high bori turnover (eg. excessive collagen in urine samples) a person with a thyroid condition such as hyperthyroidism; a person who has experienced a fracture after only mid trauma; a 25 person who has had -ay v idence of vertebral fracture or other signs f osteoporosis As noted above, osteoporosis can aso result as a condition associated wi i another disorder or from the use of certain medications, Osteoporosis resulting from drugs or another medical condition is known as secondary osteoporosis. In a 30 condition known as Cushings disease, the excess amount of cortisod produced by the body results in osteoporosis and fractures. The mnost comnron medications - 44 associated with secondary osteoporosis are the corticosteroids, a class of drugs that act like oretisol.a hormone produced naurally t he arena glands. Althouah adequate levels of thyroid hornones (which are produced by the thyroid gland) are needed for the development, of the skeleton, exces thyroid hormone can decrease .5 bone mass over time. Antacids that contain aluminm can lead to bone loss when taken in high doses by people with kidney problems, particularly those undergoing di'aysis, Other mledications that can cause secondary osteoporosis include phenytona (Dilantin) and barbiturates that ar used to prevent seizures; methotrexate (Rheunmatrex. Inie, Folex PFS) a drug for some forms of arthritis, cancer. and 10 imnmune disorders; cyclosp)orie (Sanimmune, Neral), a drug used to treat some autoimmune diseases and to suppress the immune sysiein in organ transplant patients; luteinizing hormonereleasing hormone agonists (Lupron Zolade),used to treat prostate cancer and enomerniosis; heparin (Calciparine Liquaemin), an anticlotting medication; and cholestyamine Questran) and colestipol (Coiestid) 15 used to treat high cholesterol Bone loss resulting from cancer therapy is widey recognized and termed cancer therpy induced bone loss (CTIBL). Bone netastases can rebate cavities in the bone that may be corrected by treatment with activin ActRlla antagonists In a preferred eibodinient, activin-ActRIa antagonists; parricuary a 20 soluble AtRIJa, disclosed herein may be used in cancer patients, Patients having certain tumors (e,g prostate, breast, muntiple myelora or any tmnor causing hyperparathyroiim) are at high risk for boni loss due to tuoor-induced bone loss as well as bone ietustases and. therapeuti agents. Such parents may b with activin-AcR Ta antagonits even in the absence evidence of bone loss or 25 bone metastases, Patiets may also be monitored fo evidence of bone sS or bone metastases, and may be treated with activin~AetRIla antagonists in the event that indicators sugge st an increased risk Generally, DEXA scans are employed to assess changes in bone density while indicators of bone remodeling may be used to assess the likelihood of hone metastases, Serum markers may be monitored Bone specific 30 alkaline phsphatase (BSAI)is an enzyme that is preset in osteoblasts. Bld levels of BSAP are increased in patients with bone netastastasis ard other conditions that result in increased bone remodeling Osteocalcin and procollagen peptides are - 45 also associated with bone fornaoin and bone iastases increases in BSAP have been detected in patients with bone metastasis caused by prostate cancer, and to a lesser degree, in bone mretastases front breast cancer, Bone Morphogenetic Protein-/ (Blf-7 levels are high in prostate cancer that has metastasized to bone, but not in 5 bone metastases due to bladder, skin, liver 5 or lang cancer; Type I Carboxy-terminal telopeptide (ICTP) is a crossink tound in collagen that is formed during to the resorption of bone. Since bone is constantly being broken down and reformed, TP vili be found throughout the body.owever, at the site of bone metastasisthelevel will be significantly higher than in an area of normal bone, ICTP has been found in 10 high levels in bone metastasis due to prostate hng and breast canen Another collagen crosslink Type I N-terminal telopeptide (NTx is produced along with TP during bone turnover, The amount ofNlx is increased in bone metastasis caused by many different types of cancer including lung, prostate, and breast cancer Also, the levels of NTx increase with the progression of the bone metastasis 15 Therefore; this marker can be used to both detect metastasis as wellas measure the extent of the disease. Other markers of resorption include pyridinoiine and deoxypyridinoline. Any increase in resorption markers or markers of bone mletastases indicate the need for aetiin-ActRIla antagonist therapy in a patient Acivin-ActR-ia antagonists may be conjointly administered with other 20 pharmaceutical agents Conjoint administration may be accomplished by administration of a single coformulationby simultaneous administration or by administration at separate times. Activin-ActRlia antagonists may be particulady advantageous if administered with other bone-active agents. A patnt may benefit from conjointly receiving activin-ActRlla antagonist aind taking calcirn 25 supplenents, vitamin Dk appropriate exercise and/or in some cases, other medication. lRxanples of other medtcations include, bisphosphonates (alndronate ibandronate and risedronate) cionin, estrogens garathyrold hormone and raloxifene. The bisphosphonaes (alendronate ibanidronate arl riedronate) caicitonin, estrogens and raloxifene affect the bone remodeling cycle and are 30 classified as antinesorptive medications. Bone remodeling consists of two distinct stages: bone resorption and bone formation, Anti-resorptive medications slow or stop the bone-resorbing portion of the boneremnodeling cycle but do not slow die - 46 bone-forning portion of the cycle As a result new formation containes at a greater rate than bone resorption, and bone density may increase over time, Teriparatidea, t form of parathyroid hormone increases the rate of bone formation in the bone remodeling cycle Alendronate is proved for both the prevention (5 mg per day or 5 35 ng once a week) and treatment (10 g per day or 70 mg once a week) of postmenopausal osteoporosis Alendronate reduces bone loss, increases bone density and reducesthe risk of spine, wrist and hip fraciures, Alendronate also is approved for treatment of glucocortcoid-induced osteoporosis in men and wonen as a result of long term use of these medications (ie, prednisoe and cortisone) and for the 10 treattuent of osteoporossin nien; -Aleadrotnae plus vitanin D is approved for the treatment of osteoporosis in postnenopausa women (70 nmg once a week plus vitamin D) and for treatment to improve bone mass in Ame with osteoporosis. Ibandronate i approved for the prevention and treatment of postmenopausal osfeoporoqi Takn caS as once-anonth pill (150 mg), ibandronate should be taken IS on the sam- da each month. Tbandronate reduces bone loss increases bone density and reduces the risk of spine fractures.Rsedronate is approved for tRe prevention and treatment of posunenopausal osteoporosisTaken daily (5 nag dose) or weekly (35 mg dose or 35 mg dose with cavelum) risedronate slows bone lossincreases bone density and reduces the risk of spine and non-spine fractires. Risedronate also 20 is approved for use by men and women to prevent and/or treat glucocortioi induced osteoporosis that results from long-term use of these medications (i e. prednisone or cortisone). Calcitonin is a naturally occurring hormone involved in cacium regulation and bone metabolism, in women who are more than years beyond menopause, calcitonin slows bone lossincasesspl bone ni 25 may relieve the pain associated with bone fractures. Cadcitonin reduces the risk of spinal fracturesCaicitonin is available as an linection [50o- Pt)0 hudaily) or nasal spray (200 M daily). Estroger herap (ET)ormone therap (H T ) is , approved for the prevention o osteoporosis E h nas been shown to reduce hone Vossincrease bone density in both the spine and hip and reduce the risk of hip and spinal taetus 30 in post meion opausal ne ieed most comnonly in the -orm of a piL or skin patch that delivers a low dose of approximately 0.3 ng daily oi a standard, dose of approximately 0625 rg daily aud is effective even when isred aMer age - 47 - 70, When estrogen is taken alone, it can increase a wmian's risk of developi-ng cancer of the uterine lining (endonetr'ial cancer). To eliminate this ri healthcae prov riders prescibh the hormone progestin in combination with estroe(or replacement therapy or.- IT) for those women who have an intact uterus. ET2HT 5 relicv es menopause symptoms and has been shown to have a beneficial effdc on bone health Side effects mayn inlude vaginai bleeding; breast tenderness, mood disturbances and galbladder disease. Ralrxiefene, 60 mg a days approved for the prevention and treatment of postnenopausal osteoporosis, Ais fim a cass of drugs called Selective Eren 'Receptor Modulators (SERMs) that have been developed 10 to provide the beneficial effects of e.st'ogens without their potential disadvantages, Raloxifene increases bone nmass and reuces the risk of spine fra turea. Data are not yet available to demonstrate that ralofeb-ne can reduce the risk of hip and other non spinie fractures. Tetiparatide. a tormi of parathyroid hormone, is approved for the treatment of osteoporosis in postmenopausal women and men who are at high risk 15 for a fractureIimedication stimulates new bone foundation and significantly increases bone mineral density postnenopausal women, fracture reduction was noted in the spine, hip, foot, ribs and wist in men. fracture reduction was noted in the spine, but there were i.sufficient data to evaluate fracturereduction at other sites. Teriparatide is selfadniostered as daily injection for up to 24 months. 20 ;. harmaeutial on ~eitin In certain embodiments acdivin-ActRfla antagonists (eg, ActRila polypeptides) of the present invention are formuet witv h a pharmacerticajv acceptable carrier. For example an ActRla polyp eptid can he administered alone 25 or as a component of a pharmaceuticals iormulation (fherapeutic composition) The subject compounds may be formulated for administration in any convenient way for use in human or veerinary nediclne In certain embodiments the therapeutic method of the invention includes administering the composition sytnicaily, or locally as an implant or de vice, 30 When administered, the therapeutic composition for use in this invention is, of coursein a pyogen free physiologically acceptable form, Therapeuticaily useful - 48 agents other than the ActRila antagonists which may also optionally be included in the composidon as describe above. may be administered sinmaneousiy or sequentially with tesubject compounds (e g, ActRita polypeptides) in the methods of the invention. 5 Typically. AcRfl ans vagonists will be administered parentally arnnace nical compositions suitable for parenteral administration may comprse one or more ActRia polypeptides in combination with one or orre pharnaceutically acceptable sterile isotonic aqueous or nonaqeous schutonsm. dispersions, suspensions or emulsions, or sterile powders which may be 10 reconstituted into sterile ejctable solutions or dispersions just prior to use, which may contain antioxidants. buffrs, bacteriostats, solutes which render the fornmuation isotonic with the blood of the intended recipient or suspendmIg or thickening agents. Examples of suitable aqueous and onaqueous carriers which may be enployed in the pharmaceutical composilcns of the invention include wa t er, ethanol polyos (such as glycerol, propylene glycoI, polyethylene glycol, and the like), and suitable mixtures thereof. vegetable ol such as olive oil, and injectable organic esters, such as ethyl oleate, Proper fluidity can be 1 aintained, for example, by the us ofng materials, such as iecithin. iy tie maintenance ofthe required particio size in tho case of dispersionsand by the use of&surfactants. 20 Further, the compositan may be e tcapsklated or injected ia form for delivery to a target tissue site (e g bone) In certain embodimenis com.positions of the present invention may inchide a matrix capable of delivering oeor more therapeutic compounds (e.g, ActRIfa polypeptides) to a target issue site (e.g, bone), providing a structure for the developing ti issue and optimally capable of being 25 resorbed into the ody For example, the matrix nay provide slo w relase of the ActRila polypeptides. Such matrices may be formed of materials presently in use for other implinted medical applications. The choice of natrix material is based on bitcompatibilitbiodegradability mechanical proprties, cosmeic appearance and interface properties. The pt ieular 30 application of the subect conpositions will define the appropriate fbrmltion. Potential matrices for the compositions may be biodegradable and chemically - 49 defined calum sulfhteticaciumphosphate, hydroxyapati te, polylactic acid and polyanydrides, Other potential materials are biodegradab'e and biologically well defined, such as bone or dermal collagen. Further natrices are comprised of pure proteins or extracelahlar imatrix components, Other potential matrices are non 5 biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics Natrices may be comprised of combinations of any of the above caentioned types of material, suchks olci a ci ad yroyate or collagen and tricaiciumphosphate The bioceramics may be altered in composition, such as in calciumalninate-phosphate and processing to alter pore 10 size particle size. particle shape, and hty In certain embodimentsmethods ofthe invention can be administered for orally, eg, in the fAt of cap'tsules, cachets, pill, tablets lozenges (using a flavored basis, usually sucrose and acacia or tragacantb powders granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water 15 in-oil liquid emulsion, or as an elixir or syrup. or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like each containig a predetermined amount of an agent as an active ingredient,-An agentnay also be administered as a bolus, electuary or paste. in solid dosage fonsfr orai administration (capsules, tablets, pills dragees, 20 powdersgraines, and the like), one or more therapeutic compounds of the present invention may be mixed with one or more pbarmaceuticady acceptable barriers, such as sodium citrate or dicalcaum phoshace and no any of the foll-w -ing (I) fIllers or extenders such as starches, lactose, sucrose glucose mannitol, and/r silicio acid; (2) binders, such as, -for example, carboxyrethy'celhdlse, alginates, gelatin 25 polyvinyl pyrroiidone. sucroseandor acacia; (3) humectants, such as g(cr; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silieats, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) aborpion accelerators, such as quaternary ammonum compounds;(7) wetting agents, such as, for e-ample, cetyl alcohol and glycerol 30 monostearate; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such a tale, calcium stearate, mnaguesiu nstwrate. solid polyethylene gigcoissoium lauryl sulfate, and fixtures thereof; and (10) colorng agents- In the case of - 50 capsules, tablets and pills, the pharmaceutical compositions may also conprise buffering agents. Solid compositions of a similar type may also be employed as fillers in, soft and hard-filled gelain capsules using such recipients as lactose o. r nlk suqa, as well as high molecular weight polyethylene glycois and the like, Liquid dosage forns .for oral administration include phamaceuticallv acceptable enmulsion ~ miroemnusions,slt ,s spe s syrups,ol and elixis. to addition to the ati ingredient the liquid dosage boms inay contalnert diens commonly usd in the art, such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol isopropyl alc.Ohol ethyl carbonate, 10 ethyl acetate, benzyl alcohol. benzyl benzoate, propylene glycol i butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ oive, castor and sesameW oils)g ycerol; teuhydrofuryl alcohol, polyethylene glycols and ramy acid esters of sorbitan, and mixtures thereof Besides inert diluents, the oral cmpostons can also include adjuans such as xwting agents emulsifi;ng and iS suspending agents, sweetening, fh ,rn, coloring, perfamingsrand poevtv agents, Suspensions, in addition to the active compounds. may contain suspending agents such as ethoxylated isostearyl alcohols polyoxyethyiene soritol and sorbitan estersmicrocrystalline cellulose, alunrn metahydroxide; be ioni e 20 agar-agar and tragacanth, and mixtures thereof The compo sitions of the invention may also contain adjuvats, suclh as preservaives, wettng agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisnis may be ensured by the inclusion oivarious antibacterial and antifungal agents, for example. paraben chlorobuanol phenol 25 sorbic acid, and the li.e. it may also be desirable to include isotonic agents, such as sugars. sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form mays be brought about by the 30 It is understood that the dosage regimen will be determined by the attending physician considering various factors which modify the action of the subject - 51 compounds of the invention Qi g.' hatRi pdvppqtidei h a~uhtr include but are not limited to amount of bone weight desired to be fonnedte degree of bone density loss, the site of bone damage the condition of the dam aged bone, he patients age, sex, and diet, the severity of any disease that may he 5 contrituting to bone loss, time of adnmustration and other linil factors Optionally, the dosage May var' with the type of matrix used in the reconstitution and the types of compounds in the composition. The addition of other known growth factors to the fina composition may also affet the dosage. Progress can be monitored by periodic asessment of bone growth and/kr repair, for examplerays 10 (inchding DFXA), histomorphometie determinations and etracycline idbeling. Experiments wih mice nave demonstrated that effects of ActRia- Y on bone are detectable when the compound is dosed at inervals and amounts sufticient to achieve sea concentrations of Ov2 pghks or greater, nd serum levels of I pg/ksg or 2 pgkg or gnreate are desirable for achieve significant effects on boe density 15 and strength Although there is no indication that higher doses of ActRISeFe are undesirable due to side effects, dosing reginens may be designed to reach ,erun concentrations of between 02 and .1 pg/kg, and optionally between I and 5 pgIkg, in humans, sermnn levels of 0.2 gg/kg may be achieved with e Single dose of O1 Ing/kg or greater and serta levels of I pg-kg may be achieved with a ingle dose of 20 13 Tng/kg or greater The observed sena m halfife of the molecule i between about 20 and 30 days, substantially longer than ost F fusion proteins and ths a sustained effective samm fevl may be achieved,,for example; by dosing wit 02 0.4 mg/kg on a weekly or biweekly basis, or higher doses may be used with loger intervals between dosings For example, doses of 1n mg/kg might be used on a 23 monthly or bimonthly basis, and the effect on bone may ie sufficiently dvirab. that dosing is necessary only once every34,.5, 6, 9, 12 or more months, In certain embodiments, the present invention also provides gene therapy for the in vio production of ActRita polypeptides. Such therapy would achieve its therapeutic effect by introduction of the ActRia polynucleotide sequences into cells 30 ortissues having the disorders as listed above. Delivery of ActRIta polynueleotide sequenes can be achieved using a recoobmant expression vector such a5 a chimeric - 52 virus or a colloidal dispersion system Preferred for therapeutic delivery of Actulla polynucleotide sequences is the use of targeted liposomes, Various viral vectors which cal be utilized for gene therapy as taught herein include adenova us, herpes virus accinia or, preferably an RNA virus such as a 5 retrovitus. Preferably, theo riral vector is a derivative of a nurine or avian retrovirs Examples of retroviral vectus in which a single foreign gene can be .letedude, but are noimited to: Noloney nurine leukemia virus (MoVbu. VI lHarvey nurine sarcoma vius (HuSV) murn nnunmay tmnor virus (MuMTV and Rous Sarcoma Virus (RSV). A number ot additioal retroviral vectors can 10 incorporate multiple genes. Alt of these vectors Can transfer or incorporate a genae for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attachingfor example, a sugar, a glycobipdeor a protein Preferred targeting is accomplished by using an antibody, Those of skill in tie art will recognize that specific polynucleotide sequences can be 15 inserted into the retroviral germne or atiched to a viral envelope to allow' target specific delivery of the retroviral vector co inning tie ActRila polynucleotide, In a preferred embodiment, the vector is targeted to bone or catilage. Atermativelytissue culture cells can be directly trarnsfected with plasmids encoding the retroviral stad genes gag, pai and env, by conventional calcium 20 phosphate transfection. These ells are tic transfected with the vector plasmid con tainng the genes of interest. , The resultinag cells release te retroviral vector into the culture medium. Another targeted delivery system for ActRla polynucleotides is a colloidal dispersion system. Colloidal dispersin systems include nacomolecule complexes. 25 nanocapsuies. microspheress beads, aid lipid-based systems including oil-in-water enulsionsmicelies mixed micelles, and liposomes The preferred Colloidal system of this imvention is a iposme, iposontes are artificial embrace vehicles which are useful as delivery vehicles in vitro and in vivaR NA DNA and intact virions can be encapsulated within the aqueous interior ad be delivenid to cells in a 30 biologialky active form(tee eg. raley, et alTrends B-iochm. Sci6:77 1981). Methods for efficient gene transfer using aise vehicle, ar- known in the art. - 53 see e~gy. Mannino, et al Biotecimques, 6:6it2, 1988 Ihe composting of the fiposome is usually a cumbiuaton ittof phosholipids, usually in combination with steroids, especially cholesterol, Other phospholipids or other lipids may also be used. The physical characterisicsof iposomes depend on pH ionic strength, and 5 the preseuc of divalent coons Exaumples of ipidsc' useful in iposone productironincl de phosphatidyl compounds, such as phosphatidylycerol phosphadylcholine, phosphatidyiserine phosphatidyethanolamne, sphingoipids, cerebrosides, and gangliosides,. Illustrative phospholipids inchide egg phosphaidyleholine 10 dipalmitofphosphatidylcholine, and distearoylphosphatidyieholine The targeting of liposonmes is also possible based on. for example, organmspecicity cl speciefity rid organellespecificity and is known in the art. EXEMPLIFIC'ION The invention now ben gene.'lly described, it will be more readily understood by reference to the flowing examples, which are induded me rdy for purposes of illustration of certain enbodiments and embodiments oft:e present invention. and are nrot intended to hnnit the invention. 20 Appicants constructed a solible AtRla fusion protein that has the extraceflidar domain of human ActRUa fused to a human or mouse Fc domain with a nmininmal linkr in between. The costructs are rferred to as ActRifahiE and ActRlnsiac, respectively . ActllHa-h4 is shown belowas purified from CHO cell lines (SEQ TD NO: 2$ 7): 2 S 7S ILOGR 81EC 1PFNAVNW EK-D RT N QTG VE P CY GDfKIDK R RHCP A TWKN ISc S I EIVKQGCW LDDINCYDRTDC VE K KDSPB VYRF CCCTNMCNEKPSYF PEME V TQPTSN PV'TPKPPTOGOTHTCPPPAPELLGOPSVFLFPPKPKDTLMSTPEV TICVVVDVSHEDPE VKFN WYVDOVEVHNAKTKP'REEQYN STYRV VSVLTVLe 30 SQN}QALCK PVPiETIKAKPRPVYTLPSREEMTK - 54 - The Ac ti-he and A m prote ins were expressed in CHO) cell iles Three different leader sequences were considered: 5 (i)Hloney bee mellitin (UIBM): M KFJ VNLVFMVVYISYIYA (SEQ UD NO: 8) (Hi) Tissue Piasminogen Activator (TIPA)- MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO, 9) (iii) Native: MAAAKLSAFAVTLISCSSCA (SEQ ID NO: 10) 10 The seeted form employs the TPA leader and has the flowing unprocessed amino acid sequence MDAMKRCLCCVLLLCOAVEVSPGAALORSETQEC.LFNANWEKDRITNQT OVEPCYGDKDIKRRCFATWKN ISGSIEVKQCCWLDDINCYDT)RTD CVEKKD SPEVYFCCCEThN~MCN EKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPA 15 PELJPSVFLPP KPKDTMISRTPEVTCVVV VSHEOPEVKFNWYVDOVE VIHNAKTKPREEQYNSTY RVVS VLTVLHQD WLNGiKEYKCKVSNK3ALPVPIE KTISKAKOQPREPQ VYILPSIZEE IKNQVSLTCLYKGFY1PSDIA VEIWESNG0 QPENNYKTIPPVLDSDGSFFLYSKT VDKSR WQQINVFSC SVMEALHNH YTQKSLSLSPiGK (SEQ i) NO:)13 20 This poIypepdde is encoded by the following nucleic acid sequence: ATG GATOC AAkTGAAGAGAGGCTCTCTTGCTCTOC0-ATOTOGAO CAGTCTT'COTTTOCCCOCGiCCGiCTATACTTGGTAGATC.AGAAACTCAO GATTTTTTATO ATGGAAGCAACATAAAC 25 0TA CCTGGOLAAGAATATTTCTOITCCATGAATAGTAAACAAGCTTGVTT GGCTGOATOA TATCAACTGTATGACAOGACTGATTOTCTAGAAAAAAA AGACA.0CCCTGAAOTATATTTCTUTTTQCtTOAGCAA~~TTiTTAATO AAAACTTTTCITTATTTTCCGGAGATOGAAGTCACACA OCCCACI TCAAAT 30 CGTCCGACGATCGGGACTA~rCTTCC - 55 - CCAAA, ACCCAAi-GGACACCCTrCAT,'GAT'(,.CCCCGG,-ACCCCTIGA(3GTC,AAT GCGTGGTGGTGGACGTOAGCCACGAAGACCGTGAGGCAAGTTCAACTG GTACGTGGACGGCGTCACGOTOCGATAATCCCAAGxACAAAGCC GCC'CGA OGACAGTACAACAGCACGTACCOTGOTCACGTYICCTCACCGTCCTG 5 CACCAG.ACTQGCTGAATGGCAAGGAGTACAAGTGCAAOGTC'CCAACA AAGCCCTCCCAGTCCCCATFCGACGAAiAACCATFCTCCAAAGICCAAA,1 ,GGGtCA GCCCCAGAA.CCCAGGTGC.(ACCT'.-(j-dCCCCK(3'"-CC- .CCGGGAGGAGAT1"G-1 COAAGACCAGOT'fI'CGCCTGACCTOCCFGTCAAGCTCA.C GCGACATCGCCO'TGGA&GGGAGAGCAI'~GCCAGCCC$CiA3.ACAACT 10 AA A A ZGT T AT G G tITTN ,in AGCAA CCTCA CC GTGGACAAGAG CA GGTGOCA (G AG GGAAC G~TYC CFG CTCCGTGA TGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAA~'ATGA0AATTC (SEQ IDI NOd4)t Both ActR~n-hFc and ActR~la-mfc were remarkably amenable to 15 recombinant expression; As shown in figure 1, the protein was ptwifed as a single, well -defined peak of protein. Nn-terinal sequencing revealed a single sequence of ILGRSTQE (SEQ ID NO: 11I Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, penylisepharose 20 chromatography, size exclusion chromatography. and cation eange chronatography The purification could be completed wih viral filtration and buffer exchange. he ActRila-h-c protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE, 25 ActRila-hFc and ActRlav-nF showed a high aIffity for ligands particularly activin A, GDF-li or Activin A ("ActA") were imrnobilized on a Bmacore CM 5 chip using standard amine coupling procedure. ActRila-hFc and AczRl~ia.-m~c proteins were loaded ono the system, and binding was measured ActRfla-hec bound to activin with a dissociation constant (K)) of Sx10- and the 30 protein bound to GDF I with a K, of 996x 0- See figure 2. ActRIiam-nEC behaved similarly. - 56 - An A-204 Reporter Gene Assay iwa used to evaluate the efficts of ActRlla hlFe proteins on sigaling by GDF- 1. and Activin A Cell line: Human Rhabdomyosarcotna (derived fom muscle) Reporter vector: pL3(CAGA)12 (Described in Deriner et at, 1998, MBO 17: 3091-3100.) See Figure >. The 5 CAGA12m present in TGFLBeta resAponve genes ( PAI gene) So this vector is of general use for factors signaling through Sna2 and 3. Day 1: Split A204 celLs into 48-well plate lay 2A204 cefls transfeoted with 10 pg p0L3(CAGA)12 or pG3L3(CAGA)12 (0 gg)y+ pt (411g) and Fagene. i) Day 3: Add factors (dinted into mediuani+01 % B3A) Inhibitors need to be pirienhaated with Factors tor I hre betre adding to cells 6 hrs later cells rined with P.BS and lyse cells. This is follow ed by a [uciferase assay, Typically in this assay.in the absence of any inhibitors, t iviusA \hows roughly 10 told stimulation of reporter 15 gene expression and an ED50 ~ ng/nl! GDF- 16 fold stimulation, E D5:VSt ng/mil (GDE-Sshows an Tfet simiar to GDKFI 1 As shown in figure 4. Ac RQa h~e and ActRIlanmEc inhibit GDF~8 mediated signaingat piconmolar concentrate. As shown -in figure 5, three different preparations of AetRla-hEc inhibited GDF41. signaling with an IC50 of 20 approxiiately 200 pM. The ActIila-hFc was very stable in pharmacokinec studies. Rats were dosed with 1 mg/kg3 mgkg or 10 mgakg of ActRna-hF protein and plasma levels of the protein were measured at 24 4 72 144 andI 1 hoturs. Ir a sepnlare studOy rats were dosed at!mg/kg 1t gk or 3 0 im In ats ActRla-hFc had an i 25 14 day serum half life and circulatheg levels of the drug wire quite high afer two weeks (11 pghnld 1 p/mIl or 304 pg/m or initial admitrations of I wg/kg 10 mg;kg or 30 mg/g respectvwly) In oynomolgus monkeys he plasma hr Itlfe was sulbstantiallygreater than 14 days and circulating levels of the dru were 25 pgml, 304 pglmi or 1440 p/ml f-r InitIal af dminitratioIn ot ng/kg 10 mg/ikg or 3( 30 mg/Ig respectively Prelininary re slts in humans suggests tha the serum half life is betven about 20 and 30 days. - 57 - REample2: ot1?Jagicgrogoes Bo ot'mtb In iv Normal female mice (ALB/) were dosed with ActRIla-nFc at a level if 1 gkgdose, mg/kg/dose or 10 mg/kg/dose with doses given twice wkly Bone 5 mineral density and bone meral content were t y DEXA, see figure 6. n BALB/t female mice D5XA scans showed a significant increase ('20%) in none mineral density and content as a result of' ActRUP -m~E treatment See figures 7 and 8 Thus, antagonism of ActRIta caused increased bone density and content in 10 normal female mice As a next step, the efct of ActRIIa-mFc on bone inamouse model for osteoporosis was tested. Anderson et al, (200) established that ovarectorized trce suffered substantial bone loss (rugiy 50% loss of trabecrdar bone six weeks post-Operation), and that bone loss in these mSer could be carcted with dIdate therapeutic 15 agents, such as parathyroid hormone, Applicants used C57BL6 female mice that were ovariectom ized (OVX) or sam operated at 4-5 weeks of age, Eght Weeks after surgery treatment with ActRPHm Pc(10 mgkg, twie wedky) or control (PBS) was vitiated. Bone density was measured by 's*rcanner, 20 As shown in figure 9, treated ovariectomized mice showed substantial loss oftrabecular bone density relative to the sham controls after six weeks ActRUa-m~c treatment restored bone density to the level of the sham operated nice. At 6 and 12 weeks of te treant ActRa-me caused substantial increase in trabecular bone of OVX aee, See figure I After 6 weeks of treatment bone 25 density increased by 24% lative to PBS controls, After 12 wek the increase was 27%, In the sham operated nice AetR UIa-me aso caused a substantial increase in trabecular bone See figure II. After 6 and 12 weeks, the treatment produced a 35% increase relative to controls - 58 in an additional set of experiments ovariectoiied (OX) or sham operated mice as described above were treated with ActRlIar'Fc (10 mgkg, twice weekly) or control (PBS) over twelve weeks, Similar to the results described above tor ActRilainFc OVX mice receiving ActRilamFc exhibited an increase in irabecular $ bone density of 1%by as early as four weeks and 25% anfer 12 weeks of treatment (Figure 12). Sham operated mice receiving ActRnta-mFc similarly showed an increasein trabeclar bone density of 22% by as early as tour weeks and of 32% after 12 weeks of treatment (F igure 13). After twelve weeks of treatment -with AtRIaan~c. whole body and ex vivo 10 femurEXA anysis showed that reatnient induces an increase in bone density in both wvariectomized and sharn operated mice (Figures 14A and 14B, respectively Ihese results are also supported by ex vivo pQCT analysis of the femoi midshaft which demonstrated a significant increase in both total and cortical bane densiy after twelve weeks of treatments with AXuRila md . Vehicle-treated control 15 ovariectomed mice exhibited bone densies that were comparable to vehicle treated control ami operated mice (Figre I5)l in addition to bone desitv, bone content increased following ActRalFniF teatment, Ex vivo pQCT analysis of the temoral midsabu demonstrated a significant increase in both total and cortical, bone content after twelve weeks of treatment with ActS.la-nt. while both 20 ovariectonaized and shan operated vehicle controtreted mice exhibitecd comparable bone content (Figure 16), Ex vivo pQCT aalysis of the femoral midshaft also showed that ActRila-nnc treated mice did not show a change in periosteal circumnference; however .Acita-mvFc treatment rested In a decrease in endosteal circumference indicating an increase in cortical thickness due to growth on 25 the inner surface of the femnur (Figure 17). Mechanical testing of thmurs deternmed thar Act~ia aas able to increase the extrinsic characteristics of the bone (mi mal load, stiffess and energy to break) which contributed to a significant increase in the intrinsic properties (ultimate strength) of the bones. Ovariectonriized i-ne treatexrd with ActRiamFc 30 exhibited increased bone strength to levels beyond sham operated, vehicle heated controls, indicating a complete mversal of th osteoporotic phenotype (igure 18 - 59 - These data demonsTate that an ac ivu-ActRIla antagonist can increase bone density in normal female mice aid, firtherunore correct defects in bone deisiy bone content, and ultimately hone strength in a mouse model of os'teoporosiN In a further set of experments, mice were ovariectomizd or sham pera td 5 at 4 weeks, ad beginning at 12 weeks received either placebo or ActR wlc (2 timneweek, 10mg/kg) (also refredm to as RAP-11 in Figures 19~24), for afrther period of 12 weeks A Variety of bone parameters were evacuated As shown in Figure 19 ActRia-mFc increased vertebrae trabecular bone volume to toi l volume ratios (B3V/T"V) in both the OVX and SHAM operated mice. ActRila mFe also 10 improved the trabecular architecture (Figure 20), increased cortial thickness (Figure 21) and improved bone sengtmigure 22 As shown in Figure 23, ActRiln-mFe produced desirable effects at a range of doses from 1mg/kg to 10 mgkg; Bone histomorphonetry was conducted at a 2 weetime point in sham operated micea These data presented in Figure 24, denmoninstrate that ActRlamE 15 has a dual effect, both inhibimtng bone rmeption and promoting bone growth. Tlius ActRIt~ff i sinmulates bone growthb(anaboltic effect) and inhibits bone resortion (anti eatabolie effect)iitsbu EAample lternatIl AcTrd a* - i Prot 20 An alternative construct may have a deletion of the C-erminal tail(the final 15 amino 'acids of the extracellular donain ot AttRia. The sequence for such a construct is presented below (Fe portion undei ned)(SQ ID NO:12): LLORSETQECLFNNM WEDiUNQTGEP \ YGDKDKRRHC FAT IWNLSSI EIVIKQCOWI DDINt2YDRTDCVEKKDSPEVY FCC CEGNMCNEKFSYFPEN'ITG 25 GOTHuPPCPAPELGPSVELPM 'IliKD"LM SRTEVTCVVVDVSNEDPEV KEN WYVDGVEkITNAKTKPREEQYNSTYRVSVI.TVLMH 1ENGKE{(C _IY S P P L Q E V RWOQGNVFS CS -MHE---NT -- YTQKSSLSPOJK 360 - 60 - INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by rtrence in their entirety as if each indiviual p ication or patent was specifically and individually indicated to be incorporated by refterice. 5 While spectfic embo d of the subject matter have been discussed the above specfication is illustrative and not restrictive. Many variations vill bcom apparent to those skilled in the art upon review of this specification and the clims below. '1e 1t l scope of the inveron irshould be determined by refreniee to the claims, along with. their full scope of equivale.nts, and the specifcation-iong, iwit 10 such variations - 61 -

Claims (14)

1. 2. The activin-inding ActR Ha polypeptide of chim 1, herein the po lypeptide is at least 95% pureo, with respect to protein contamnants as determined by size exclusion chromaiography 3 The activin-binding ActRffa polypeptide of dis i or 2,whein the polypeptide eahibis least 10-id selectivity in dissociation conslantor atain verns3 CDP 11 10 4 A pharmaceutcal preparation con mprisin the activin-binding ActIla polypeptie of any of claims d3 and a pharmacenucaIly acceptable excipient. 5, The phnrmuceuticai preparatio of claim 4, wherein said prepuratiofn is substantialy pyrogen INree 15 6. An isolated polyucleotide coprising a coding sequence for the activin binding AetlUsa polypeptide of any of clauns l3 7, The isolated potide of o-aan 6 in the isolated potide comprises asequence of SEQ ID NO 14, 8 A recombinant placleodde comprising a promoter sequence operably 20 linked to a polynuceotidc of claims 6(o 7; 9 A cell transforned vith a recomBiant polyuu lectide ofay of claims 6- 8 1.0. The cell of ain 9 wherein the I mammalian oelv, 11, The call of claim 10,berein the cell is a CH0 cal or a human cell. 1.2. A method ormaking an activin-binding ActRla polypeptide, comprising 25 a) culturing a cell under conditions suitable for expression of thesoluble ActRita polypeptide, wherein said cel is transformed with a recombinant polyucleotide of any of claims 6-8; and ) recovering the activinbinding Act Ria polypeptide so expressed. - 62 - 11 A method for promoting bone growth.increa ng bone dnsh or mtirg bote Strength, the method comprising administering to a subject an effective amount of a polypeptide selected from the groUp COns of: a) a polypeptide omprisign anino acid sequence ac east 90% identical to 5 SEQ &D NO2 b) a polypeptide comprising an amino acid sequence at least 90.identicdto SEQ ID NO:3; and c) a poypepude comprising at Last 50 consecutive amino acids selected from SfEQ JTD NO 2 10 14. The method or clm 13, wherein polypeptide has one or mom of the following charactesuis i) biids to an ActRTIa lgand wih a K. of at least 10 M, ; and i) inhibits ActRla signalhing a cell,
2. 15. T he rethod of claims I3 ir 14, wherein said polypeptide is a fusion protem 1 in hzindg, in addition to an ActRufa p pde domai one ir mo-c polypeptide portions that enhance one or more of in veto srabifv, n vivo half f uptake'admn istradot, tissue iocaliation or distriution, formation of protein-" comiplexes and/or pu rification
3. 16. The method of claim 15 wherein said fusion protein includes a polypeptide 20 portion selected fron the group consisting of an inmnunogkobdin Fc domain and a serimi aibunin., 17, The method of any of aims 13-16 ,wherein said polypeptide includes one or m-ore modified aneino acid residues selected from: a glycosylated amino acid, a PEoylated amin acid, a farnesylated amino acid, an acetylaten amino 25 acid, a biotinylated amino acd, an mino acid con jugated to a lipid moiety, and an amiio acid conjugated to an organic derivatizing agent, 18, A method for treating a hone-related disorder the nehed comprising administering, to a subject in need thereof, an effective amount ofa a ivin or ActRIa antagonist. - 63 -
4. 19. The method of claim 18. wherein the activin or ActRlla antagonist is an activin or AcitIa antagonist polypeptide. 20, The method of claii 19, wherein thc actnivn or ActRila antagonist polypepide is selected from the group casisting of: a a, polypeptide comprising an an-ino acid sequence at least 90% identical to SEQ fD NO: 2 b) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:3; and c) a polypepid. comjrising at least 50 consecutive amino acids selected 10 from SEQ ID NO: 2 21, The method of claims 19 or 20, where the activin or Actita antag st poTypepide has oTe or more of the following charactersucs; i) binds to an ActRIla ligand with a K 0 of at least 10, tM; and ii) inhibits ActRlla signaling in a cel 15 22. The method of any of claims 19-21, wherein said activin or Actikla antagonist polypeptide is a fusion protein inekding, in addition to an ActRiua polypeptide domain one or more polypeptide pot ions that enhance one ormore of in vivo stability n vivo half life uptak/administrain 4 tissue localhzatem or distributionf ation of protein. complexes and/or 20 purificatior
5. 23. The metod of claim 22, wherein said fusion protein includes a poiypeptide portion selected famnt the group consisting of an im unogLulin Fc domain and a serum albunin
6. 24. The method of any of claims 19-23, wherein said activin or ActRila 25 antagonist polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PRGyiated amino acid a farnesylated amino acid., an. acetylated amino acid, a biorinyiated amino acid, an amimo acwid conjugated to alipid moIety and an amino acid conjugated to an organic dervatizing agent. - 64 - The method of any of claims 18-24, wherein the bonere[aed disorder is selected from the group consisting of: primary osteoporosis and secondary osteoporosis, 26, The method of any of claims 18-24, wherein the bone-rmelated disorder is 5 selected fOttn the group casistfing of post mnopausal kOsteOrs hypogonWaa bonelosse tumor-induced bone loss, cancer therapy induced bone loss, ony, metastases nmtpie omydoma and Paget's disease
7. 27. The method of any cfclaims 18 26 wheimn the method further comprising admiistering a second bcne-active agent 10 28. The method of clam 27, wherein the Lonefaetive agent is selected from the group consisting of: a bisphosphonate, an estrogen, a selective estrogen receptor modulator, a parathyroid hormone, a caictonn a caleAnn supplement and a vitaminD supplement
8. 29. A hannaceuticai preparation com prisinag IS (a activin or ActRla antagonist; and (b)a second bonesactive agent, 30 A metd of identifying an agent that promotes bone growth or inc reasez bone density the method comprising a) idenftfing a test agent that binds to a ligandbinding doinain of an 20 ActRiUa polypeptide competitively with an activin or ActaRIla antagonist polypeptide; and blevaiating the effect of the agent on growth of the tissue, 1 Usc of an activhi or ActRila antagonist polypeptide for snaking a medicament fior the treatment of a boneerelated disorder. 25 32 A method for prevening a bone-related disorder, te method cornmpnri sing adini;texing, to a subject in need thereof an effective amount of an activin or ActRIla antagonist 33 The method of claim 32,; wherein the subject has a cancer that is associated with bane metastases - 65 - 34, The method of claims 3233, wherein the subject is positive for an indicator of loss of bone density, bone resorpton, or bOne metastases 35 The method of any of claims 3234, wherein the subject is the recipient of cancer treatment regimen that is associated with bone los 5 36. The mehod of any of claims 3244, wherein the subject has a cancer associated~ whwh bne loss 37, ''he activin bindhig ActRIiapopepoid f f any of claims 1-3 the pharmaceucal preparation ofan of lams 4~5 or 29, or the method of any of claims 13-28, wherein the polypeptide is glycosLxated, 10 38, A method for promoting bone growth and inhibiing bone resorption in a patient, the method comprising administering to the patient an effective amount of a ActR11a~FE fbsion protein, wherein the ActRUla-o fusion protein. comprises an amino acid seqence that is at least 90% identical to the amino acid sequence of SEQ IDNO:3, 15 39. The method of claim 38. wherein the ActUla.e fusion protein comp rises an amino nid sequence that is at least 95identical to the amino acid sequence of SEQ 0 iNO3.
9. 40. The method of claims 38 or 39, wherein th Act(0aFe fusion potein composes the amino acid sequence of SEQ ID NOI 20 41. The method of any of claims 38-40, where ein he AtRIia-Fl fusion protein coinpnses the amino acid sequene \SeQ ID NO 2
10. 42. The method of any of laiins 38-41 wherein the nihod causes less than 10% increase in the patient's setal mRuscle mas 4' The method of any of clainis A-41, wherein the ActR.laF fusion protein is 2 adinistered so as to reach a serum concenin ii he patient of at least 02
11. 44. The Method of any of clainrs 3843, wherein the ActRia-c fusion protein has an amino acid sequence of SEQ ID NO:7 - 66 -
12. 45. The method of any of cla-ns 3844 wherein the ActRia-Fe fusion protein has a serun haffdif of between 5 and 30 days.
13. 46. The method of any of claims 38~45, herein the ActRIa-Fc fusion proteit is administered to the patient no more frequently than once per week 47 "The method of any of claims 38-46, wherein the ActRIac fusion protein is adniinistered to toe patient no more frequently than once per month, 48, Use of a polypeptide se5ected from the group consisting of: (a) a polpeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2; (b) a polypeptide comrising an amio acid sequence at least 90% identical 10 to SEQ ID NO and (c) a polypeptide comprising at least 50 consecutive aino aids selected from SEQ iD NO 2 for the preparation of a medicament for promodig bone growthincreasing bone density or increasing bone strength. 49, se of an activin o Act.Rita antagonist polypeptide for the preparation or a 15 medicament for preventing a bone-reflted disorder. 50, Use of an ActRIaffil fusion rotei comprising an amino acid seguence that is at least 90% identical to the amino acid -squence of SEQ ID NO: for the preparation of a medicament for promoting bone growth and inhibiting bone resorption. 20 5.1 A polypeptide selected from. the group consisting ofa a poiypeptide comprising an amino acid sequence at least 90% iden.tica to SEQ ID NO:2 (b) a polypetide comprising an amino acid sequence at least 90% identical to SEQ UD NO:3 and (c) a pivpeptide comprising at least 50 consecutive aminio acids soelecd froum SEQ D NO: 2, for use in promoting bone growth. 25 increasing bone decnsty or micreasing bone strength. 52 An activin or ActRila antagonkst polypeptide tfr use in the treatment of a bone-related disorder
14. 53. An activin or ActRIla antagonist polypeptide for use in preventing a bone related disorder. - 67 - $4, An ActRila .J-e fu.~ion omei -omlprisinig an amino -ac-id euneta is at least 90% identical to the amino acid sequence of SEQ ID NO:3 for use in promoting bore growth and inhibitig bone resorption. 10 - 68 -