AU2015200950B2 - ActRII receptor polypeptides, methods and compositions - Google Patents

Abstract

In certain aspects, the present invention provides compositions and methods for modulating (promoting or inhibiting) growth of a tissue, such as bone, cartilage, muscle, fat, and/or neuron. The present invention also provides methods of screening compounds that modulate activity of an ActRII protein and/or an ActRII ligand. The compositions and methods provided herein are useful in treating diseases associated with abnormal activity of an ActRII protein and/or an ActRII ligand.

Description

ACTRII RECEPTOR POLYPEPTIDES, METHODS AND COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of Australian Application No. 2012244215, which is incorporated in its entirety herein by reference.

This application claims the benefit of U.S. Provisional Application Serial No. 60/590,765, filed July 23, 2004. All the teachings of the above-referenced application is incorporated herein by reference.

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.

The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two general branches: the BMP/GDF and the TGF-beta/Activin/BMPIO branches, whose members have diverse, often complementary effects. By manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in an organism. For example, the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass.

Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Changes in muscle, bone, cartilage and other tissues may be achieved by agonizing or antagonizing signaling that is mediated by an appropriate TGF-beta family

In certain aspects, the present disclosure provides ActRII polypeptides. Such ActRII polypeptides may be used for the treatment of a variety of disorders or conditions, in particular, muscle and neuromuscular disorders (e.g., muscular dys&amp;<J|>i$r, smyofeophio lateral sclerosis (ADS), and muscle atrophy), undesired bone/eartilage growth, adipose dssue disorders (e,g,, obesity}, metabolic disorders (e,g., type 2 diabetes), and neufodegeneforiVe1 disorders. Ih bpeeifift embodiidents, ActRII polypeptides; (e,gf, soluble^^i^ly'piijHdeS) fcan antagonise an ActRII mebpfoh.(b<g·, ActRIIA or AetRIIB),generally, in sny process: associated with ActRII activity, Qptfonallyi ActRII polypeptides of the invention may lie designed to prelemntiaHy antagonize one dr mote ligands of AetRli; receptors, snob as GOES (also called myostatin), ©DF11, activist Modal, and BMP? (also called OP-1 ), and may therefore be useful in foe treatment of additional disorders. Examples pf ActRIf polypeptides include, the naturally occurring ActRIf polypeptides as well as fonctional varianis thereof.

In certain aspects, the disclosure; provides phunmaceutieiilprepmutidns comprising t soluble ActRII (e.gn ActRUA or ActRIIB) polypeptide that binds to an ActRII ligisnd such as GBFS, G0F11, aetiyin, BMP? or nodal, and a pharmaoendoaUy

Optionally, the soluble ActRII polypeptide binds to an ActRII ligand with a Kd less than 10 micromolar or fess tban 1 micromolar, 100,10 or l «anonfolar. Optionally, the soluble ActRII polypeptide inhibits ActRII signaling, such as hdracellnkr signal transduction events triggered by an ActRII ligand, A soluble ActRII polypeptide for use In such a preparation maybe any of those disclosed .herein, such as a polypeptide having an amino acid sequence selected from $RQ ID NOs; 1*2 and 9-12, or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 92%: or 99% identical to an amsno acid seqaenee selected ftom SEQ ID MQs: 1*2 and 9*12. A soluble ActRII polypeptide may include a functional fragment of a natural ActRII polypeptide, such as one comprising at least 10, 20 or 30 amino adds of a sequence selected iiorn SEQ IDfolDs: 1 -4 and 9-12 or a sequence of SEQ: ID NOs: l or 2, lacking the Cbtermina! 10 to IS mnlno adds (the A soluble ActRII polypeptide may

Include one of more alterations in the amino acid sequence (e.g., in the llgandfoinding domain); relative to a naturally occurring ActRII polypeptide. The: alteration in the amino acid sequence may, for example, alter glycosylatiop of fee pblypeptide when produced in a mammalian^ insect or other eukaryotic cell or alter proteolytic cleavage of fee: polypeptide relative fo the naturally occurring ActRII polypeptide,: A soluble ActRII polypeptide may be a fusionprotein that has. as one domain, an ActRIf polypeptide (e.g., a bgand-binding domain of an ActRII) and one or more additional domains feat provide a desirable property, such as irngfoved pharmacokinetics, easier purificatimp: targeting: to: particular tissues, etc.

For example, a domain pl:a fusion protein may eulmneeone or mom oFih vivo stehility, in vivo tissue loealfeation or distributioBsfeimaiion of protein complexes, muibmeriMtion of the fusion protein, end/or ponficstion. A stdnbfe AetRII fusion protein may include an immunoglobulin Fe domain (wild-typeor mutant or a serum albumin, in a preferred embodiment, an AeiRfRFc fusion comprises a relatively anstmetored linker poAtipned between the Fc domain and the extracellular AclRii domain. This unstructured linker may conesppnd to the roughly 15 amino acid: unstructured region at the ip-terminal end of the extracellular domain of ActRlIA or AetRIlB (the “tail”), or it may be an artificial sequence of between 5 and 15,20,30, 50 or more: amino acids that are relatively free of secondary structure. A Unker may be rich In glycine and proline residues and may, for; example, contain repeating sequences of threonine/serine and glycines (e.g., TG* or SO4 repeats^ A fosion protein; may include a purification subsequence, such as an epitope tag, a FtlA0 tag, a poiylnstldine sequence, and a GST fusion, Optionally, a soluble AetRII polypeptide includes one or more modified amino acid residues selected from: a: glycosylated anrinp acid, aFBGylated amino acid, a fkmesylated amino apid, an acetylated amino acid, a biotinylated amino acid, an ammo acid coigngated to a lipid moiety, andan amino acid conjugated to an organic derivsfizing agent. A pharmaceutical preparation may also include one or more additional compounds such as a compound that is used to treat asr ActRIF associated disorder. Preferably, a phannaeemica! preparation is substantially pyangeuiree.

Ip general, it fe preferable thatan ActRii protein be expressed in a mammalian cell line that mediates suitably natural glyeosylation of the AetRII protein so as to diminish the likelihood of an unfavorable immune response in a patient. Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression veefom will be useful

In certain aspects, the disclosure provides packaged pharmaceuticals eoniprising a pharm aceutical preparation described herein and labeled for use in promoting growth of a tissue or diminishing or preventing a loss of a tissue in a human. Exemplary tissues include bone, cartilage, muscle, fat, and neuron.

In certain aspects, the disclosure provides soluble ActRii polypeptides comprising an altered ligand-binding (e.g,, GDF8~bpdmg); domain of sn AetRIL Such altered ligand-binding domains of'an AetRII receptor compriseone or more mutalions at: amino aqid residues such as E3?, E39, ROO, R5S, RS6, Y60, A64, K74, W?R L79, D80, F82 and F10I of human

AetRIIB, Suchaltered ligand-binding doxnaihs of an ActRII receptor comprise one or mote mutations at amino acid residues such as E38> E40* R4l, KS6, RS7, Ytil, Kj55, K7S, W79, : L80> 081, '183 and F102 of human ActRIIA, Optioxmllyythe altered ligand-binding doxnain can have increased:selectivity for a ligand such as GDF8/GDF1 1 relative to a wiidAype ligand-bsndingdemain of an ActRII reeeptext To illustmie, these roatatious are demonstrated herein to increase the selectivity of the altered ligand-binding domain for GDF! 1 {and therefore, presumably, OOFS) over aetivin (pfoiforited with respect to ActRllB); 1C74Y, K74F* K74I and D80I:-, The following the ratio of aetMo binding over GDFΠ: DS4A, IC55A, L79A and F82A. The overall {GDFll and actiyin) binding activity can be increased by inclusion Of the “fair repon or, presnnphly, a «Hstruemred linker region, and also by use of a mutation such as Ati4R (which occurs naturally) of R74A> Other mutations that caused an overall decrease in ligand binding affinity, include- R4i)A, E37A, E56A, W78A, D80.R, D80A, D80G, D80F, D80M and B8fM. Mutations may be combined to achieve desired effects. For example, many of the mutations that affect the ratio of GDFll rActran binding have an overall negative effect on ligand binding, and therefore, these may be combined with, mutations that generally increase ligand binding to produce an improved binding protein with ligand selectivity.

Optionally, the: altered iigand>bMding domain has a, ratio of IQ for aetiysn binding to IQ for GDF8 binding; that is at least 2, 5, It), or even 100 fold greater relative to the ratio for tire wild-type. hgmrdfoxudffig domain. Optionally, the altered ligand-binding domain has a ratio of ICs6 for mMhiilng xrelrvin to iCjsa^dnhibifihgjGDFS/GDFl 1 that is at least 2,5,10, or even 100 fold greater relative to the wild-type ligand-binding domain. Optionally, the altered lipnd-binding domaln inhibits GDFB/GDFl I with an.ICso at least 2,5,10, or even 100 times less than the iCjo'fot inhibiting; activia, These soluble ActRII poly-peptides can be fusion proteins that xnchxdean immunoglohulm Fc domain (either wild-type or mutant), in certain: cases, the subject; soluble AetMI. polypeptides are antagomsts flrdubitars) of GDFS/GDFil,

In cepain aspects, the disclosure provides nudeie acids encoding a soluble ActRII polypeptide, which do not encode a complete ActRII polypeptide. An isolated polynucleotide xnay comprise a coding sequence for a soluble ActRIIpolypeptide, such as described above. Fur example, an isolated nucleic acid may include a sequence coding for an extracellular domain (e.g., ligand-binding domain) of an ActRII and a sequence that would code for part or aB of the transmembrane domain and/or fJie oftopiasoric domam of an ActRII, but for a stop eodoo positioned within die transmembtane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated pafynucf eotide'mag «S>iact|pH«#· glbIMe«$Eh

ActISli poSynacieoiide sequence: such as/SEQ; ID NCR 7 or 8, or a partially truncated version, said isolated polynucleotide Igriber comprising a transcription hundred nucleotides before the d’rierminas or otherwise positioned: such ^ the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a full-length AetRXL Nucleic acids disclosed herein may be operably li nked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides. Preferably the. ceil is a mammalian: :cell such as a: CHO oeli.

In certain aspects, the disclosure previdi^mbiMid$ fpf taking $;«61uble Aetlll polypeptide. Such a method may .Include expressing any of the nucleic acids (e.g., SEQ ID NO; 5 or d) disclosed herein in a:suitable cell, such as a Chinese hamster ovary (CIHOf cell. Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble ActRII poly'poptide, wherein said cell is transformed .with., a soluble ActRI! expression construed and b) recovering the soluble ActRfl polypeptide so expressed, Soluble ActM! polypeptides may be recovered as crude, paAially purified or hi^rly purified tractions using any of the well known techniques lor obtaining protein from cell cultures.

In certain aspects, a soluble ActRII polypeptide disclosed herein may be used in a method for treapng a sufyeot having a disorder associated with muscle loss or msafficleut muscle growth. Such disorders Include muscle atrophy, muscular dystrophy, amyotrophic lateral sclerosis fAFS), and a muscle wasting disorder (e g,, cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophies, neuromuscular diseases, motor neuron diseases, diseases of the neuromuscular junction, and inflammatory myppathiesg A method may comprise administering to a subject in need thereof an effective amount of a soluble ActRII. polypeptide. in certain aspects, a soluble ActRII polypeptide disclosed herein may be used in a methodifbr treating a subject having a disorder associated with neurodegeheration. Such disorders include Alzheimer’s Disease (AD), Parkmsori’s Disease. (FD), Amyoimphie katefal Sclerosis (ALS), Huntington % disease (HP). A meiiod1 may comprise administering to a subject in need thereof an effective amount of a soluble ActRII polypeptide.

In certain aspects, a soluble AciRII polypeptide disclosed herein may be used in a method for beating a subject having- a disorder assoeiated with abnormal coil gmwih and differentiation, Such disorders include inflammation, allergy, aatohmhune diseases, infeetious diseases, and tumors, A method may comprise administering to a sabieet in need thereof an effective amount of a soluble ActRH polypeptide, A selective aetivtn binding AetRII protein may beparticularly useful for treating an aciiyin-dependsnt cancer, such as ovarian cancer.

In certain aspects* a soluble AotMI polypeptide disclosed herein may fee used in a method for decreasing the body fat content or reducing tire rate of increase in body fat content, and for treating a disorder associated with tmdesirahle body weight gain, such as obesity, non-insulin dependent diabetes meiiitus (NIDBM), cardiovascular disease, cancer, hypertension, osteoarthritis, stroke, respiratory problems, and gall bladder disease. Ihese methods may comprise administering to a subject In need thereof an effective amount of a soluble ActRH polypeptide.

In certain specific aspects, a soluble AotEII polypeptide disclosed herein may be used in a method for heating a disorder associated with abnormal activity of 013F8. Snch disorders: include metabolic disorders such as ..type 2 diabetes, impaired glucose tolerance, metabolic· syndrome, (β,-g·, syndrome X), and insulin resistance induced by trauma (e.g,, bams or nitrogen imbalance); adipose tissue disorders (e,g., obesity); muscular dystrophy (including D«ehenne?s muscular dystrophy); anryo:^ lateral sclerosis (ALS); muscle atrophy; organ atrophy; haifty;:: carpal tunnel syndrome; congestive obstructive pulmonary disease; ssreopenla, cachexia and other muscle wasting syndromes; osteoporosis; glueoeonicoid-mdueed osteoporosis; osteopenia; osteoarthritis; osteoporosis-related iraoinms; low hone mmstiue io bhronic glucocorticoid therapy, premature gonadal Mime, andmgeh suppression, vitamin D deficiency, secondary hyperparathytoidisin, nuttifiboal deficiencies, ^;anore3tiat:::mfvos&amp;. Ibe method may comprise admmisteriog to a subject in need thereof an effective amount of a soluble AetEII polypeptide.

In certain aspects, the disclosure provides a method for identifying an agent that stimulates growth of a tissue such as bone, cartilage, muscle, fit, and neuron. The method comprises; a) identifying a test agent that binds to a ligand-bindmg domain of an ActRll polypeptide competitively with a soluble ActRH polypeptide; and h) evaluating the effect of the agent a® growth of the tissue.

In certain aspects, the disclosure provides methods for antagonizing activity of an ActRIl polypeptide φ an ActRIf ligand (e.g., GDFfo GOFI 1, activin, BMP7, and Nodal) in: a cell The methods comprise contacting the ceil with a soluble ActRIl. polypeptide.

Optionally ,: the activity of fee ActRIl polypeptide or fee ActRIl ligand is: monitored by a signaling transduction mediated by the AetRIl/AetRlI ligand complex, for example, by monitoring cbll prolifeTation, The ceils of fee methods Include an osteoblast, a chondrocyte*, a myocytey an adipocyte, a ntuscle cell, and a neuronal cell

IncerialnaSpectss the disclosure provides uses of a soluble ActRIl polypeptide for making a medicament ,for the treatment of a disorder or pondiiimt as described herein.

Brief Ocscrintion of the Drawings

Figure 1 shows a human ActRIIA soluble (extracellular} polypeptide sequence (SEQ ID NO: I). The C-tenninal. “tail” is underlined.

Figure 2 shows a human ActRIIB soluble (extracellular) polypeptide sequence (SEQ ID NO: 2), The CAerminal ‘'tail” is undetlincd·

Figure 3 slfowa hiunau ActRIIA precursor protein sequence (SEQ ID NO: 3). The the extracellular domain is in bold (also referred to as SEQ IP NO: j); and the potential Nfenked glycosyiation sites are boxed.

Figure 4 shows human ActRIIB precursor protein sequence (SEQ ID NO: 4). The Signal peptide is underlined; the extracellular domain is in bold (also referred to as SEQ IP NO: 2); ahd the potential M-lmked glycosyiation sites are boxed.

Figure 5 shows a nucleic aoid sequence encoding a human AefRlIA soluble (exiraeelfelar) polypeptide, designed as SEQ IP NO: 5.

Figure d shows a nucleic acid sequence encoding a human ActRIIB soluble (exiraeel&amp;fep pblypeptide, designed ha EEQ IP NO: 6.

Figure 7: shows a nucleic acid sequence encoding fmpen ActRIIA preeurspr protein, designed as SEQ ID NO: 7.

Figure $ Shows anueleic acid: Sequence encoding human ActRIIB precursor protein, designed as SEQ ID NO: 8.

Figure? $ shows expression of the extmceiiular (soluble) domains of ActRIIA or AetRJlS. Constniets expressing human extracellular domaists of AciftllA or ActRXiB were made with all three signal sequences.

Figure 10 shows three soluble AetRHB polypeptides with various sijpal seqaeuces, SE QlDNOs'A-lL

Figure 11 shows one soluhle AetBIjA polypeptide with its n&amp;dwsignal sequence, SEQ IB MC): 12.

Figure 12 shows design ofthe Fc fissions of AetRIi A or ActRllB polypeptides. The flexible linker sequence and the Fc sequence (SEQ ID NO: 13) am shown, Maiafiohs can he made at one more amino acid residues of the Fe sequenee, ixamples of such residues for mutations are underlined, and relfeped to as Asp-2b3, lysine-322, and Asn-434.

Figure 13 shows the ligand-binding pocket of an AefRlIB polypeptidei Examples of amino acid residues in the ligand-binding pocket are shown as B39, K5$, Y60;, K74, W7S, 030¾ and F I 01. ActRIIB polypeptides of the tnyendna may comprise mutations at one or more of these amino apid residues.

Figure 14 shows an alignment of the extmeellular domains of ActlillA and AetlGIB, with the positions of mutations that, in ActRIIB, are demonsMted herein to affect ligand binding. The alignment shows that the position of these mutations is conserved in ActRIIA

Figure I S shows a schematic fer the A-204 Reporter Ciene Assay- 'H»e figure shows the Reporter yeetor: pOD3(CAGA)12 {described ia Dennlegetai, Ij^SkEMBO- Π: 3091-3100.) The GAGA12 motif is present in TGF-Beta responsive genes (PARI gene), so tips vector is of general, use ft*-factors signaling through SmadS and 3,

Figure 16 shows the effects of various mutations in ActRlIB-Fc on a BDF-l l A-204 Reporter Gone Assay. Tire background A64 construct showed the least effect on GDF-11 activity. The A64R mutation (also a naturally occurring /form). caused a substantial increase in GDF-11 ^inhibition, and a combination of the :Ad4K mutation with the addition of the 15 C~ terminal amino acids of the extracellular domain (the 1S ammo acid “taif'') produced an even more potent inhibitor of G BF~ 1! acti vity.

Figure 17 shows the: effects: of-various mutations in ActRBB-Fc on an Activln A, A-204 Reporter Gene A&amp;say. The background A64 construct showed the least effect oh Aetivih A: activity, The K74A mutation caused a ««bstaniisl increase in AhtiviuA inhibition, A control sample lacking Actiyin A showed no activity.

Detailed Description of the- Invention I, Overview

Ik certain aspects, tbe present mventioh relatesto ActK.II polypeptides. As used herein, the term '‘Act-RID refers to a family of aetiviu receptor type II (AetRII) proteins and AetRII-related proteins, derived feom any species* Reference to AetRII herein is understood tobe a teference to any one of the cunenriy idsnrified fijrms, including A<hRB A (also known as AetRII) and ActKIiB* pemhers of the AetRII family are genemlly all transmembrane proteins, composed of a ltgand-hmding extracellular domain with eysteineufeh region, a transmembrane domain, and a cytoplasmic domain ^with jn^iefed serine/tbmonme kinase specificity. Amino acid sequences of baman AciRIlA precursor protein and ActMIB precursor protein are illustrated in Figure 3 (SBQ ID NO: 3) and Figure 4 (SBQ ID NO: 4), respectively.

The term. “AetRII polypeptide*’ is used to refer to polypeptides comprising any naturally occu,tTingpol^eptide of an ActRU&amp;mily member as well as any variants thereof (including ittuiautS, fusions. and peptidomimetic forms) that retain a useful activity. For example, AetRII polypeptides include polypeptides derived from fee sequence of any known AetRB haying a sequence at least about 80% identical to the sequence; of ast AotRli polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity, lb a specific embodiment, the invention mlates As

described herein, the term “soluble AetRII po1ypepride!5 generally ;mfemtp pohfpepildcs comprising an exbnceliular domain of at» AetRII ptefem. The term %ohdde AetRII polypeptide,” as osed herein, includes any naturally occurring extmeellular domain of an AetRII protein as well as any variants themof (including mutants, imgnmnts and peptidomimetic ferms) that retain a .useful activity. For example, dre extracellular domain of an AciRII protein hinds to a ligand and is gene rally soluble. Examples of soluble AeiSlI polypeptides include ActRIlA and ActRIIB: soluble polypeptides illuArated in Figure 1 (SEQ ID NO: 1) and Figure S: (SEQ ID NO: 2), respectively. Other examples of soluble AetRB polypeptides comprise a signal sequence in addition to the extracellular domain of an AetRII protein, for example, the 10 (SB0 ID N0s: 941} and Figure 11 (SEQ ID MO: 12). Tile signal sequence can fee a native signal sequence of an ActBIk or a signal sequence from another profom, sbeh as a iissne plasminogen activator (TP A) signal sequence or a honey Me rnelatin (HEM) signal sequence. TGF~p signals am mediated % lieieionierio eomplexesofi^»; 1 and type 11 serine/ threonine kinase receptors, which phosphotylaieand activate downstream Smad proteins upon ligand stimulation (Massages, 2000, Mat. Beu, Mol. Cell EM. 1469-178). These type I and type II receptors are all tensmsmhmne pmteins, eotnpdsed of a l lgaad-binding extracellular domain with cysteine-rich region, a tmesmembrune domain, and a cytoplasmic domain with predieted serine/tlireonine specificity. Type 1 receptors am essettdal for sighaling; and type II receptors are expmssibn of type 1 receptors. Type I and 1.1 aedvin receptors form a 'stable complex after ligand binding, msuldngipphp^hdiylation of type 1 receptors by type II receptors. TWO related type II receptors, ActRHA Piid' AchfidlB, have -beep identified as the type II receptors for aetivins (Mathews and Vale, 1991, Geli 66:973-982; Afosanoet ah, 1992,

Geli 68; 97-108), Besides adivia^ ActMlA and AetEIIB eaa biochemically interact with several other ΤΘΕ-β family proteins, laciading BMP?, Modal, 0DF8. and G DPI l (Yamashita el a!,, 1995, T GcIi Biol. 130:217-226; tee and McPherron, 2001, Proc, Mat!. Acad, Sci. 98:9306-931ly Ybo and Whitman, 2001, Mol. Cell 7; 949-957; Oh et a!., 2002, Genes Dev. 16:2749-54).

In certain enfoodiments, the present in vention relates to antagonizing a ligand of AetEli. receptors (also referred to as an AetBlI ligand) with a subject Actfoil polypeptide (e;g,, a sdl able Actfoli polypeptide). Thus, compositions and methods of the; present invention are usefol for treating disorders associated with abnormal activity of one or more ligands of ActRIl receptors. Exemptary ligands of AetMl receptors include some TGF~p family rnernhera, such as aetlvia, Modal, ODES, GDP11, and BMF7, These ligauds of ActEIl receptors are described in more detail below..

Aetivins are dimeric .polypeptide growth factors and belong: to foe YGF-beta seperfenrily, There are three aetivins (A, B, and AB) that arc homofoeterodimers of two closely related p subumf s (fApA, papa, andpApg}, In tire TOEdmm superfemily, aetivins are unique and multifunctional factors that emt stimulate hormone production in ovarian and placental cells, support neuronal cell survival, inBuenee eellteycle progress positively or negsrivefy depending m cell type, and induce oiesodenual difeeuliatlott at least in • {^Paolb ef-.al..> 1991, Ptse SecEp Biol Med, 1.98:500-51¾ Dyson etal, 1997, Curr Biol. 7:81-84;.Woodruff, 199¾BioehemPhannaeoi 55:953-90), Moreover, erytfarpid diffemniiahorr tltotor (EOF) ’frbt^=:.ll3&amp;;'^iExi^]EilaiMed [liuman monocytic lenlcemie cells was fouM to be Identical to action A /Mpmta et ah, 1988, PNAS, 85:2434). it was suggested thafaetivin A acts ad atiatoml regulator of eiyttiiopoiesis in toe bene toa prow. In several tissue^, aetivin: signaling is antagonized; by its .related heterodimer, htoibin, For:eaample,:d«ring tbe :release of folltefc-stimulating bonotme (FSH) from toe pituitary, aetivin promotes FSH secretion and synthesis, while inhibln prevents FSH sectetiop and syjdhem Other proteins that may regelate aetivin bioaeiivity and/or bind to aetivin inclnde tollistadn (FS)i tollistatto-tslated protein (F'SRP), a^mmeroglobulm, Cerberus, and endoghn, which: are described below,.

Nodal proteins have functions in mesoderm and endOderhi induction and formation, as well as subsequent organization of axial structores :SUch as heart and stomach in early embryogenesi#, It has been demonstrated that dorsal tissue in a developing vertebrate embryo contributes predominantly to the axial stmefores of tbe notdebwd andpfo-ehq>rdal plate while it recruits surrounding cells to form nem-axial embryonic structures. Nodal appears to signal through both type 1 and type 11 receptors and intracellular etTeetors Mown as Smad proteins, Recent studies support the idea foat ActRIIA: and ActRIJB serve as type II ei ah, Genes Cells, 2002,7:40142). It is suggested ihafNodal ligands intensci with their codactors (e.g,, eripto) to activate aetivin type I and type II receptors, svhleh pbosphorylate Smad2. Nodal proteins are implicated in many events critical to the early vertebrate embryo,: Including mesoderm:: .fbmtation, anterior patterning, and left-right axis speesrieation, Experintenial evidence has demonstrated that Nodal signaling activates pASd-Lnx, a luclferuse reporter previously shown to respond specifically to aetivin and TGF~bcta, Howe ver^ Nodal is unable to induce plla24,uxs a reporter speciticaily mspensive to heme morphogenetic proteins. Recent results provide direct biochemical evidence that Nodal signaling Is Mediated by both aedvfo-TGF-beta pathway Smads, Smad2 and Sntadl, Further evidence has shewn that die^ extracellular cripto protein is required for Nodal signaling, making it distinct from aetivin or TGF-beia signaling.

Growth aud Oifferentiatioa Factor-8 (GDF 8} is also known as myostaiia. GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in the developing artel adult skeletal muscle, lire GDFS null motatioo in immgenlo rnlce is-characterized by a marked hypertrophy and hypoplasia of-the skeletal muscle· (MePhmon etal, Nature, 1997, 387:83-90). Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF8 in cattle (Ashmore et al>: l 9:74, Growth, 38:501 »507; Swatland and •Kiefiir» 7. Anim, ScL 1904, 38:752-757; MoPbcrron and Lee, Proa. Natl Acad. Sci USA, ! 997s; 94; 12457-1246.1; ^tobador ^ah^Genome Sos., 1997,7:910-915) and* strikingly, in humans (Sehuelke et al,D' Engl 1 Med 2004;3S<12682~8):. Studies have also.shown that muscle wasting associated with HW-mfoetlen in humans is accompanied by increases in GDF8 protein expression(Gotmalez-Csdayid etal., PN^S, 1998,95:14938-43). 1»addition, GDF8 cap modulate the ppductiou dftnuscle-speciiiprihzyrpes (e,g.s creatine Mpase) and modulate myohkst cell prohforahan (4FO 90/43781). ileGDFS propeptide can noneovalently hind to the mature GPF8 domain dimer, Inactivating its hlofogleal activity (Miyaxono.et al. (1988) J. Biol Chern,, 263: 6407-6415; Wakefield et si {1988) 7. BM. Chem.,263; 7646-7654; (199O):0w?»%:^fe»S, 3:35-43). Other proteins which bind to GDF8 or structurally related proteins and inhibit their biological activ«y include follistatifo et al (1999) Bev. Biol, 298:222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP! 1, is a secreted pfofekt(MePhsrron etal, 1999,Flat. Genet 22:260-264). GDF11 is expressed in the tail bud. hush had, maxillary and mandibular arches, and dorsal root ganglia during mouse developmerd (Nakashima etal, 1999, Mech. Bev, 80: 185-189). GDF11 plays a /unique, role in patterning both mesodermal and neural tissues (Gamer et al,, 1999, Dev Biol., 208:222-32). GDF11 was shown to be a negative regulator of chondrogenesis and myogenesis in developing chick hmb (Gamer et al, 2001, Bee Biol. 229:407-20). The expression of GOBI1 in muscle also suggests its role in regulating muscle growth in a similar way to GDF8. In addition, the expression of GBFiJ iu brain suggests that GBF11 may also possess activih® Oiat relate to the function of Ore nervous system. Interestingly, GDF11 was found to inhibit neurogenesis in the olfactory epithelium (Wu et al, 2003, Heuron, 37:197-207), Hence, GBFO may have In vitro and in vivo applications in the treatment of diseases such as muscle diseases and neumdegeserative diseases (e.g,, amyotrophic lateral sclerosis).

Bone morphogenetic protein {BMF7)f also called osteogenic groteinA (0Ϊ-Ι), is well known to indaee cartilage and bone formation. In addition, BMB7 regulates a wide array of physiological processes, For example, BMP? .pay- be the osteoinductive factor responsibte for the phenomenon-of epithelial osteogenesis, it is also f%md that BMP? plays a role in calcium regulation. and bone homeostasis. Like actmn, BMP? binds to type II receptors, AetElIA and SB. However, BMP? and activin recruit distinct type I receptors into beteromerie receptor eoMplexes, The major EMF7 type I receptor observed was AUC2,. while aetlvh bound 'exclusively to ALK4 (ActRIIB). BMP? and aotivin elicited distinct biological responses and activated different Smad pathways (Mseias-Silmetal, 1998,1 Biol Chem, 271:25620-36}.

In. certain aspects, the present Invention relates to the use of pertain AcCRl I polypeptides (mg., soluble ActRil polypeptides} to antagonize AotElI receptors generally, in any process associated with ActRil activity, Optionally, ActRU pol^pttd^of^mventtrna: may antagonize one or more ligands of AetElI receptors, such as aebvitt, Nodal, ODES* GDFll, and BMP?, and may therefore be useful in the treatment of additional disorders.

Tliereibre, the pmsent inyention contemplates using AetMI polypeptides in treating or pre venting diseases or conditions that are associated: with abnormal activity of an ActRU or an ActRU ligand. ActRU or AetRII ligands am involved in the regulation of many critical biological processes. Due to their key functions in these processes,, they may be desirable targets for therapeutic iiniervention., For example, AetRII polypeptidesLe,g., e.g,, soluble ActRU polypeptides) may be used to treat human or animal disorders or conditions. Example of such disorders or conditions include, but are not limited to, metabolic disorders such as type % diabetes, impaired; glucose tolerance, meiaboKe syndrome fe.g, , syndrome X), and insulin resistanoe induced by trauma (e.g., bums or nitrogen imbalance); adipose tissue disorders (e.g., obesity); muscle and neuromuscular disorders such as muscular dystrophy (including Dnchenne ’s muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscle atrophy; oj^an atrophy; frailty; ea^al fennel syndrome; congestive obstructive pulmonary disease; and satcopema, cachexia and other muscle wasting syndromes. Other examples nfelude osfeoppmsis, especially in the elderly and/or postmenopausal women; gluedcortieoid* fcdueed osteoporosis; osteopenia; osteoarthritis; and osteoporosis-refated fractals.: W further examples include low bone mass due to chronic glucocorticoid therapy, premature gonadal failure» androg# suppression, vitaiom D deficiency, secondary hype^amthyroidism, mitritioaai deffeieneies, and anorexia nervosa, These disorders and conditions are discussed below nnder”Bxemp|aiy Therapeutic Uses,”

The terms used in this specification generally have their ordinary meanings In the art, within the context of this: invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the in vention and how to make and use them, The scope or meaning of any use of a term wiii be apparent from the specific cofttext in which the term is used, "About” and "approximately” shall generally mean an acceptable degree of error for; the/quantity measured given the nature or precision of the measurements, Tvpiciffy, exemplary degrees of error are within 20 percent (%)>preferably within 1.0%, andmare pmfembty within 5% of a given value; or range of values.

Alternatively, and particularly in biological systems, the terms"about” and may mean values that are within an order of magnitude, padhrahly within ;;S~ fold and more preferably within 2-fbid of a given value, Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about” or "approximately” can be irifeppd when not expressly stated.

The methods of the invention may include steps of comparing sequences to each Other, including wild-type sequence to one or mote mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e,g., using sequence alignment programs andferalgorithms that etc well known: in the art;(for example, BLAST, FASTA and MBGALK3N, to name a few). The skilled artisan eaoreadliy appreciate that, in such alignments, where a mutation Contains: a tesiduc/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, "Homologous,” in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evoluti onary origin,” Including proteins from supeffetrriiies in the same species of organism, as well-as homologous potfeins from different species of organism. Such proteins (and their encoding nucleic acids) haws sequence homology, as reflected by their sequence -similarity, whether In terms of percent identity or By the presence of specific residues or motifs and conserved positions.

The -sequence similarity,’’ in all its gramma tical forms, refers to the degree of identity or coirespondsoee between nucleic acid or amino acid sequences that stay or may not share a common evolutionary origin.

However, in common usage and in the instem application, theferm GmmoIogeuss!:' when modified· with so adverb such as "highly,’* may reforto sequence similarity and may or may tmi relate to a ooinmosr evotmionary origin, 2. ActRH Polvoeptldes

Ih certain aspects, the invention relates to ActRIl polypeptides (e.g,, solnlsle AetRil polypeptides), Etefotabiy, the fragments, fenctiohai variants, and modified forms have similar or the same biological activities of their corresponding wild-type ActRIl polypeptides. For example, ad^ctllOjxjJypeptide of the ipvemiprs may hind to and inhibit fhwtson of an. ActRliprotem and/or an AetRII ligand protein (e.g,, activm, Nodal, GDF8, GDP11 or BMP?), Optipnal|y, ' aP AetKE polypeptide modulates gro wth of tissues, such as bone, cartilage, .muscle, fat, and/or neuron, Examples of ActRIl polypeptides includehuman ActMI A precursor polypeptide {SEQ ID HO: 3), human ActRHB precursor polypeptide (SEQ ICs NO: 4), soluble human AetRIlA polypeptides (e.g., SEQ 3D MOa: 1 and 12), soluble human AetRHB polypeptides (e,g., SEQ IB NOs; 2 and 9-11). in pertain embodiments^ isolated fragments of the ActRIl polypeptides can he obtained by screemug polypeptides recomhinantly produced from the corresponding fragment of the nucleic acid encoding an ActRIl polypeptide (e>g., one ofSBQ IDNOs: 1-2 end 9-12), In addition, Eagmer# can he cbemically synihesmed using techniques known ip the art such as conventional Merririeid solid phase f-Mbe or t-Roc chemistry, The fragments can.be produced (recomhinanily or by chemical synthesis) and tested to identify those peptidy! fragments that can ifbnction, for example, as antagonists {inhibitors) or agonists (activators) of an ActRIl pfotein or an ActRIl ligand.

Id eertam embodiments,: a functional variant of the AetRH polypeptides has an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID bfOs: 1-2 and 9-12. In eerfaiu cases, the iunetional variant has an ammo acid sequence at least 10%, 8S%, 90%, 95%, 97%, 9S%, 99% or 100% identical to an amino acid sequence selected from SEQ ID 140s: 1-2 and 9» 12.

In certain embodiment, the present invention contemplates isaking fiaKtionsl variant by modifying the stracture of an ActEll polypeptide for such ptirposes as enltaneing tltempeetic efficacy, or stability' fe.g,, ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified ActMl polypeptides when designed to retain at least one activity of the namraily-occnn-ing form of the ActRII polypeptides,; are considered functional equivalents of the nammlt v-occurring AciRU polypeptides. Modified AefEXI polypeptides can also he produced, for instance,, by amino acid substitution, deletion, or addition, For instance, it is reasonable to expeel that an isolated replacement Of a leucine with an isoleneine or valine, an aspartate with a glutamate, a threonine with a sefine, of a similar replacement of an amino acid with a structurally related atmuo acid (e-g., eohservative mutations) will not have a mmor efieetcra the biplpgical activity Of the resulting molecule, Cfonservafive replacements are those that take place within a family of amino acids that are related hi their side chains. Whether a change in the amino acid sequence of an ActRII polypeptide results in a iunetional homolog can he readily determined by assessing the ability of the variant ActRII polypeptide to produce a response in cells In a jhshlon similar to the wild-type ActRI! polypeptide. in certain specific embodiments, the present invention contemplates making mutations in the extracellular domain (also referred to as ligand-binding domain) of an that '^:ν^ρί(όρρ»ί8ηΐ) AetRIl polypeptide lias altered ligand-binding activities (e.g., binding aMuity or binding specificity), In certain cases, such variant AeiRIX polypeptides have altered (elevated or reduced) binding affinity for a specific ligand. In other cases, the variant ActRII polypeptides have altered binding specificity lor their ligands.

For example, the variant AefRIl polypeptide pmfemhfislly binds to a specific ligand (eqp, OOFS). For example, amino acid residues of the ActRII 8 protein, such as E39, K55, YdO, 1C74, W78, D80, and FI01 (shown in Figure 13), am In the ligand-binding pocket and mediate binding to Its ligands such as aodvin and <3DP§. .Urns, 'the/pmsentlnyenfion. provides an altered ligand-binding domain (e.g., pDF8~btnding:dot#in)tof an: ActRII receptor, which comprises one or more mutations at those amino: acid residues. Optionally, the altered ligand-binding domain can. have increased selectiyiy for a ligand such as Gf)F8 relative to; a wild-type llgand'-binding domain of an ActRII receptor. To illustrate., these mutations increase the selectivity of the altered ligand-binding domain for GPRS over activin. Optkmaily, the sliered KgaM^iidmg domain has a mile of-K&amp;ibr activinbinding to fox' GDF8 feisdiag tBat Is at least 2,.-5, it), or even 180 fold greater relative to the ratio for die wild-type ligand-binding domain, Optionally, the altered ligand-binding domain has a ratio of 1C» tor inhibding; aetivin to 1¾ for inhihitiog G0FS that is at least 2, S. I0> or even 100 Ibid greater relative id the wild-typo ligand-binding domain, Optionallys the alteted wibcsn ICso at least 2,5,19, or even 100 times less than the !C$o for inhibiting activist.

As an specific example, the posidvefy-ohatged amino acid residue Asp (I>80) of the iigand-biftding domairi of AetMlB can be mutated to a different amino acid residue such that the variant AetM poiypepbde^^binds to GDF8, bat not activin. Preferably, the .060 residue is changed to an amino acid residue selected from the group consisting of: a uncharged amino acid residue,a negative amioo acid residue, and a hydrophobic amino acid residue. As will fee recognized by one of skill in the art, most of the described mutations, variants or modifications may be made at the nucleic acid level or, in some eases, by post translational modificabOn dr efsimical synfhesis, Such techniques are weii known in the art

In certain embodiments, thepresent invention contemplates specific maiahons of the AciRil polypeptides so as to alter the giyeosyiatiou of the polypeptide, Exemplary glycosylation sites m AetMlA and AciltOB polypeptides am illustrated in Figures 3 and 4 respectively:. Such mutations may be seieeted m as m Introduce or eliminate one or:more glycosylation sites, sueh as Ci-linked or N-Bhked glycosyiatioh sites, Asparagine-lmked glycosyiatbn recognition sites generally comprise a tripeptide sequence, a$paragine~Xr threorune (wbem ‘'Xs' is any amino acid) which is specifically meognizsd by appropriate ceilular glycosylation enzymes. The alteration may also be made by tire addition of, or substitution by, one or more serine dr threonine residues to the sequence of the wild-type ActRJi polypepide (lor Odinhed glycosylation sites), A variety Of amino acid substitutions or deletions at one or both of the first or lltird amino acid positions of a glyepsylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on an ActRll polypeptide is by chemical or enzymatic coupling of glycosides to the ActRll polypeptide. Depending on the coupling mode used, the sugarfs) may he attached to (a) arginine and histidine; φ) hoe carboxyl groups; (¢) free sulfhydryl groups such as those of cysteine; (d) free hydroxy! groups such as those of serine, threonine. or aromatic residues such as those ofphenylalanine, iyM&amp;iuet or tryptophan; or (f) the anode group of glutamine. These soedrods aro described in WO S7/d5330 pubUshed Sep, 1 ί, 1987, and in. Aplin and Wriston (iMl) €RC Crll Rev, Bioohem,,pp. 259-306, incorporated by reference herein. Removal of one or more: carbohydrate moieties present on an AetRli polypeptide inay be accomplished chemically and/or enzymatically, Chemical degly cdsylation may involve, for example, exposure of the AetRll polypeptide to the compound triduoroniethanesulibhic aoicl> or an equivalent: compound, This treatment results in the cleavage of most or all sugars except the finking.; sugar(N-aectylglueosamine: or N-'acetylgalaeidsamino}* whdtrlea ving'the amino acid sequence intact. Chemical deglycosylatipn is briber deseribM by Hskimuddm etal. (1987) Arch. Riochem. Biophys, 259:52 and by Edge et ah (1981) Anal. Biochem, 118:131. Enzymatic cleavage: of caAohydrate moieties on AetRll polypeptides can be achieved by the use of a variety of endo~ and pxo-glycosidases as described by Thotaknra et al. (! 987): 2detli-Enxymol, 138:350, The sequence of an AetRll polypeptide stay he adjusted, as appropriate, depending on the type of expression system used, as mantmallans yeast, insect and plant ceils may all introduce differing giycosylation patterns that can bo affected by the amino add sequence of the peptide. In general, AetRll proteins far use ® humans will fee expressed in a mammalian cell line that provides proper glyeosyktloOs such as HEK293m€fR3 coif lines, although oilier mammalian expression cell lines are expected to be useful as well.

This dl$clps^''^|bbt;:p0hbemplates a method of generating muiams, particularly sets of combinatorial mutants of an AetRll polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful R^.:^hi^inf,fhnetional variant sequences. The purpose of screening snub combinatorial libraries may be to generate, for example, AetRll 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 below, and such assays may be used to evaluate variants. For example, an AetRll polypeptide variant may he screened for ability to bind to art AetRll polypephde^ to prevent binding of an AetRll ligand to an AetRll polypeptide.

The activity of an AetRll polypeptide or its variants may also be tested in a cell-based or in vivo assay. For example, the effect of an AetRll polypeptide variant on the expression of genes involved in bone production in an osteoblast or precursor may be assessed. This may, as needed, be performed in the presence of one or more recombinant AetRll ligand protein (e.g., BMP'?}, and cells may be transfected so as to produce an ActRH polypeptide and/or variants thereof, and optionally, an AcfRH ligand, Likewise, an AeiRlI polypeptide may Be adnrinisteted to a mouse or other aomtai and one or more Bone progeriies, such as density or volume may Be assessed, The healing rate ior bone fiactnres may also Bp evaluated, Similarly, fee activity of an ActRII polypeptide or .its variants may be tested in muscle eels, adipocytes, mid neuron eels ior any effect on growth of these cells, ibr example, by lie assays as described below, .Such assays are well known and routine in the art,

Cornbinatorially^derived variants can be generated which have a selective potency relative to a ,natnraly occurring Acifelt polypeptide. Such, variant proteins, when expressed from tecombinanf BNA constructs, can be used «1 gene therapy protocols. Likewise, mutagenesis can give rise to variants which have intracell alar half-lives dramatically different than itbe corresponding a wild-type AcSH polypeptide. For example, tBe altered protein can be rendered either mom stable or less stable to proteolytic degradation or other cellular processes which result in destruction of,, or otherwise inactivation of a nati ve ActRII polypeptide. Such variants, and the genes which encode them, can Be urilixed to alter ActRif polypeptide levels by modulating thp:^lf4ife'df:'^AdeUI:p0lypepti^, For instance, a short: half-life can give rise to more manstent bidlogioai effects and, when part of an Indnethle ex|5ression symem, can allow tighter eontrni of recombinant ActRII polypeptide levels within the cel.

In a preferred'embodiment, the combinatorial library is produced By way of a degenerate library of genes encoding a homy of polypeptides which each include at least a portion of potential ActRII polypeptide sequences. For instance, a mixture of synthetic oligonucleotides can be enaymaticaly ligated into gene sequences such fiwt the degenerate set of potential ActRII polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins: (e,g., for phage display).

There arc many ways by which the librnry of potential homologs can Be generated irom a degenerate Oligonucleotide sequence, Chemical synthesis of a degenerate gene sequence can be carried out in an automatic "DMA synthesiser, and the synthetic genes then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art (see lor examine, Narang, SA (1183} Tetrahedron 39:3; Iteknra et al., (IBS 1) Recombinant DNA, Proc, 3rd Cleveland Syropos.

Maeromok.cu!e% ed, AG Waite®, Αιηδ^!®φ»«κ .l^evier pp2? ; Itafcura et ah, ($>84) Anne. Rev. Blochem, 53:323; Jtakum el:41,, (1984) Science 198:1056; Iks Mai, (1083) Nucleic Acid Res. 11::477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scon et al, (1990) Science 249:386-390; Roberts eta!., (1992) FNAS USA 89:2429-2433; Devlin etaf, (1990) Science 249:404MO6; Cwidtfeui, (1990) FNAS USA 8f: 6378-6382;:as: well as IhS,.patent Nos: 5,223,409,5,198,346, and 5,096,815s.

Alternatively, other forms of mate genesis can be utilized to generate a combinatorial library. Fox example,, ActRIl polypeptide variants (both agonist and antagonist forms) can be generated and isolated fro® a library by screening using, for -example, alanine scanning mutagenesis ami the like (Ruf eial,, 0994) Biocbemistry 33:1565“l572; Wang et al., (1994) h Biol, Chens. 269:3095-3099; Balint etal, 11993) Gene 137:109-118; Grodberg et at, (1993) Eur, J. Biochem. 218:597-601; Nagashima et al, (1993) J. Biol Chem. 268:2888- 2892; Lowmaft ct al, (1991) Biochemistry 30:10832- H>838; and Cunningham etal. (1989) Science 244:1081-1085), by linker scatteing mutageftesis {Gwstin et ah, (1993) Mixology 193:653-660; Btowh et al., (1992) Mol.. Cell Biol. 12:2644-2652; McKnight et al, (1982) Science 232:316); by saturation mutagenesis (Meyers etak, (1986) Science 232:613); by PCR metagenesis (Leung et al,, (1989) Method Cell Mol Biol 1: .11-19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller ei al,, (1992) A Short Course in Bacterial Generics, CSHL Press, Cold Spring Harbor, Mf) and Gte?4er el ah, (1994) Strategies in Mol Biol 7:32-34), Bldlserisdiwitrmg m«ta^d^.^artte«ferly In a combinaiortal setting, is an attractive method ter idebtilying truncated (biMetive) terms of AetRII polypeptides. In a methods can be used ter making soluble temta of ActRII polypeptides, which can act as agonists or antagonists of AetRII fonetions, A wide -range of techniques are knotvn in the art ter screening pne products of combinatorial libraries made by point mutations and truncations, and, for teat matter, ter screening cBNA llbrariba ter gene products having a certain property. Such techniques will be;genera|ly adapfeble fbf rapid seteenidgiof the gene libraries generated by the combinatorial mutagenesis of ActRlf polypeptides. The most widely used techniques ter: seteesing large gene libraries typicaliy conteriscs cloning the gene library into replicable exptesspn vectors, transforming appropriate cells with the resulting library of vectors, and expressing &amp;e combinatorial genes under conditions hi which detection of a desired activity facilitates relatively easy isolado» of the vector enc oding the gene whose product was detected. Each of the illustrative assays described below are amenable· to high: through-put •analysis as necessary to screen large numbers of degenerate sequences created, by combinatorial' mutagenesis techniques,

In certain embodiments, the AetRII polypeptides of the present invention include pepddomimetics. As used herein, the term “peptldomimetie” Includes chemically modified peptides and peptide-dike· molecnles that contain non-fcatesrally occurring amino acids, peptoids, and the likes I^ptidomime&amp;s provide variobs advantages over a peptide, including enhanced, smbility when, administered to &amp; subject Methods for identifying a peptidomimerie ate well known in the -art and include th e screening of databases that contain libraries of potential pepfidomimeties. For example, the Cambridge Structural Database contains a collection of greater than 300,000 compounds that have known crystal structures (Allen et ah, Acta Gcystallogr, Section B, 35:2331 (1979)). Where no crystal stmcfure of a target molecule is available, a structure can be generated using, for example, the program CONCORD (Rusinko eta!,, 3. Cbem, In! Compel Sei. 29:251 ¢1989)). Another database, the Available Chemicals· Directory (Molecular Design Limited, Informations Systems; San Leandro Calif.), contains about 100,000 compounds that are .commercially available and also 'em he-searched to identity potentia l peptldormmetks of the AetRII polypeptides.

To illustrate, by employing scanning mutagenesis to map the amino acid residues of an AetRII polypeptide which am involved in binding to another protein, peptidomimetic compounds can be. generated which mimic those residues invol ved in binding. For instance, aon-hydrolyzabie peptide analogs, of such residues can be generated using benzodiazepine (e.g., see Freidlngeret ah, to Peptides: Chemistry and Biology:, G.E. Marshall ed, BSCQM Publisher; Leiden, Netherlands,. 1988), asepine (e.g., see HuITman et at., in Peptides: Chemistry and. Biology, Gil. Marshall ed ,, ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et at, in Peptides; Chemistry1 and Biology, G.E, Marshall ed,, ESCOM 'Publisher Leiden, Netherlands, 1988), keto-me%lerte pseudopeptides (Bwenson et a!., (1986) 3. Med, Cham. 29:295; and Bwenson et at., in Peptides: Structure and fimctlon (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), b-turn dipeptide cores (Nagai et ah, (1985) Tetrahedron Lett 26:647· and Sato at at, (1986) 1 Chem Sec Perkin Trans 11231):, and h-aminoalcohois (Gordon et aL, (IfBS) Bioehem Blophys lies Common 126:4.19; and Batin et al, (1986) Bioehem Bibphys Res Common 134;?1).

In certain embodiments, the ActRil polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the ActRil polypeptides. Sttch modifications include, Inst are not limited to, acetylation, narboxylafom, glycosylafion, phosphorylation, Ilpidation, and acylation. Asa fosulfi the modified ActRil polypeptides may contain non-amino acid elements, such as polyethylene iglycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such' non-amino acid elements on the functionality of a AetRII polypeptide may be tested as described herein for other AetMl polypeptide variants. When anActRil polypeptide is produced in cells by cleaving a nascent form of the ActMSf pplypepfide, posMrmsktkmai processing may also be important; for correct folding and/or fonetfon of the protein, Diffemnt celts (such as CM), Hein, MD€i£, 29/, WBS, MIH-3T3 or HESS93) have specific cellnlar maebinery and characteristic mechanisms for such posidmnslaiional activities and may he chosen to ensure the correct modification and processing of the ActRil polypeptides.

In certain aspects, ftmetlonar variants or modified forms of foe ActIM polypeptides include iliSipn proteins having at least a pmtion of foe ActRil polypeptides and one or mom fusion domains. Well known examples of such fusion domains Include, but are not limited to, poly histidine, Giu-Glu, glutathione S transforase (GST), foiomdoxin, protein A, protein G, an immunogfobuim heavy chain constant region X$o% maltose binding protein (MBP), or human serum albumin. A fosion dornaiu nray be seleeled so as fo eonier a desired property. For example, some fission domains are paffienlarly usefol fer isolation of tint fusion proteins by affinity chromatography. For foe purpose of affinity purification, relevant matrices for affim^ ehromatopaphy, such as glntafoione-, amylase-, and nickel- or cobalt* colligated resins are used. Many of such matrices are a vailable m 4Mt” form, such as foe Pharmacia GST purification system and tire QlAexpress5M system (Giagen) useful with (111¾ fusion partners. As another example, a fosion domain may be selected so as to fiscilitate detection of ire ActRil polypeptides. Examples ofsach detection domains include the variousiluorescent proteins (e.g., OFF) as well as “epitope tags,” which are usually shot! peptide segnences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available includePI.AG, Mlueexa vims haemagghitifim (MA), and e-mye tags. In some eases, the fosion domains have a protease cleavage site, such as forItemr Xa or Thmmhife which allows the relevant potease to partially thgesf the fusion proteins ami thereby liberate the recombirmut proteins therefrom. The liberated proiems can then be isolated fem hie fusion domain by suhsequeat claomatographic separation, in certain preferred embodiment, an AeiRil polypeptide is teed with a domain that stabilizes the “stabilizer” d<mtam). By ii^bihging?> is meant anything that increases serum hall 11¾ regardless of whether this is heOapse of decreased destruction, dectesed eleatance by the kidney, or other pharoiseokuietie effect Fusions with the FC portion of an immunoglobulin are known to confer desirable pharoiacokinetic properties on a wide range of proteins, Likewise, fusions to human serum albumin can con fer desirable properties. Other types of fusion domains that may he selected include rmsltimerisrng {e.g,, dimerizing, tetramerkingf domains and fenctiona! domains fthat confer an additional biological thnetion, such as further stimulation of musele growfe)-

As a specific example, the present protein as a GDPS antagonist which comprises ah Fc domain (e,g., SEQ ID NO: 13). THTC PPC PAPSLtP3©PS¥FLFP PKFKBTLM ί S RT PEVTCVVVD (A} VSHED PSVKFNWYVD© WVHMAKTKPEEEQYN'S'fliSWSVLTVljHODSihSaiiETiCCX i A) VSNKALPVPXEKTISK&amp;K SQPREPQVYTt.PPSREBMTh^QVSLTCEVKQFyEgBjAtfSWESNGOPEMByKfTFFXljSSDg :]?ΕΕΕΥ5ΕΟΤνθΚΕΕ»00βΚ^Ρ8€8νΜΗ1ΐ4ΕΗΝ: CA) BYTQXSBteSI’OK*

Preferably, the Fc domain has such as Asp~2&amp;$, lysine 322, and Asn-434 |see Figare 12). In certain oases, the mutant Fc domain having one·· or mote of these mutations (e,g., Asp«2t>5 shniatimtjhas ieduced ability of binding to the Fey receptor relative to a wiidiype Fe domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g,, Α§η·43$ mutation) of binding to die MHf3 class Xnxdated Ec-rocepte CFeRN) relative to a wildtype Fe domain.

It is understood that different elemenis of the fusion proteins may be arranged in any mantierdte is consistent with te desired fbnctkmality, For example, an AefRlf polypeptide tnay be piaeed ^-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed O-fernrmal to an ActRii poNpeptide. The ActRH polypeptide domain and the heterologous domai n need not he a^aOent in a fusion protein, and additional domains or amino add sequences may be included O or N-termina! 10 either dbtnain or between the domains.

In certain embodiments., contain one or tnore modifications that, are capable of stabilising the ActRII polypeptides. For «sample, such niodificaiions enhance the in vitro half Hie of the ActRII polypeptides, etetanee circulatory half lift of the ActRII pol^sepiides of reducing pmte&amp;Iyiio degradation of the ActRII polypeptides, Such sfaMlteing mudiiltetioos inelnde, but are not limited .to, fusion proteins {Including, for examples fesiqn proteins comprising ae ActRII polypeptide and a 'S^ii^dbrttainV:mo4i£caHcm,of a gipPsylatis»t.:Mie. (including* for example, addition of a glyeosylation site to an ActRII polypeptide), and modifications of caftohydrate moiety (including, for example* removal of carbohydrate moieties from an ActRII polypeptide), I» the ease of ftsion proteins, an ActRII polypeptide is fused to a stabilizer domain such as an IgCI molecule (e.g,, art Fc domain). As used herein, tire term 'ustab3iz^''dkhna}n^'iKtt.:oB^r:' refers to a fusion domain (e#, Fe) as in the ease Of fusion proteins, but also includes nonproteinaceous modiScations such as a carbohydrate moiety, dr nonprotetMceous polymer, such as polyethylene glycol.

In certain emhodirnenis, the present invention makes available isolated andfer purified toons of the ActRII polypeptides·, which are isolated from, or other proteins.

In certain embodiments, ActRII polypeptides («amoditied or modified) of the invention can: be produced by a variety of artdmown techniques. For example, such ActRII using standard protein chemistry techniques such as those described inBodansky, M. Frineiples of Beptido Synthesis, Springer Verlag, Berlin (I#|) and Grant G. A, (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, Nesv York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 39¾ MilligeftBioseamh 9ΗΰΟ), Alternatively, the AetRlI polypeptides, fragments or variants tlmmof may be recnmbmantly produced using various expression systems (e.g, > B. coil, Chinese Hamster Gvary cells, COS cells, baculovtrns) as is well -Mown in the art (also see below), In a further embodiment, the· modified dr unmodified ActRil polypeptides may be produced by digestion of naturally occurring or reeombinsntly produced ftii-hmgth ActRII polypeptides by using,ibr exampie j a protease, teg,, trypsin, tbermdlysm, cbymotrypsin, pepsin, or pained basic amino: acid converting enzyme (FACE). Computer analysis (using a commercially available sofrware, teg., Mae Vector, Omega, PCGene, Molecular Simulation, lac.) can be used to identify proteolytic, cleavage sites.

Alternatively.,: such AetRII polypeptides may he produced from naturally occurring or tecombinanily piOdueed Ml-lbhgth AciRlt polypeptides sa&amp;h as standard techniques known in the art, such as byciiernical cleavage (e.g., cyanogen bromide, hydroxylaminc). hi certain aspechg hie invention provides isolated and/or recombinant nucleic acids encoding any of the ActRIl polypeptides|e.g;, soluble ActRIl^p^ypaytides}, ineMSing fragments, functional variants and fusiongroteins disclosed herein, Ftwexamjde, SEQ ID NDs: 7-8 precursor polypeptides, while SEQ ID NO's: 5*6 encode solnbl#,Ae^r^lypeptide&amp;. The subject nucleic acids may be single-vStrande-d or double stranded. Sndb ndeldc acids tday be DNA or A molecules. These nucleic acids are may be ase<l for example, in methods for making ActRIl polypeptides or as direct therapeutic agents (e.g.? in a gene therapy approach). hi certain aspects, the subject nucleic acids encodingsActRIl polypeptides are further understood to include nee!eic acids that are variants of $FQ ID N©: 7 or 8. Variant nucleotide sequences ihelude ssquenees tbat diShr by one of more nucleotide substitutions, additions or deletions, such as allelic vartmttspand will i&amp;emhsfe, iheiude coding sequences that differ from the nucleotide sequenes of the coding sequence designated in SEQ ID NO: 7 Or 8. in certain embodiments, the invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 8SM, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5 or 6. One of ordinary shill in the art will appreciate that nuc leic acid sequences complementary to SEQ ID NO: 5 or bpand variants of SEQ ID NO: '§ or 6 are also within the scope of this invention. In further embodiments, tire nucleic acidseqaences of the Invention can be isolated:, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of die invention also Include nucleotide sequences that hybridize under highly stringent conditions to:the nucleotide sequence designated in SEQ ID NO: 5 or b, complement sequence of IEQ ID ND; 5 or 6, or fragments thereof As discussed above, one of ordinary skill in the: art will understand readily that appropriate sidogency conditions which promote DNA feybridleation can be varied. One of ordinary skill in the art will understand readily that promote DNA hybridisation eats be varied, Jporexampfe, one could perform the feyferidlxation at 6J x sodium ehteride/sodinm citrate (SSG) at about 45followed by a wash of 2 (1 x SSC at 50 °C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at SO °C to a high stringency of about 0,2 x SSC at 50 °C. In addition, foe tempemtute in the wash step can he increased from low stringency conditions at room temperature, about 22 °C, to high stringency conditions at about 05 °C. Both temperature and salt may bewaried, or temperatom or salt concentration may fee held constant while the ether variable is changed, In ohe embodiment, the invention femvidos nuelcic adds which hybrid me under low; stringency conditions of 6 x SSC at mom temperature:followed by a wash: at 2 x SSC at room temperature.:

Isolated nucleic acids which differ Iron1, the nucleic acids as set forth In SEQ ID NOs: 5-6 due to degeneracy in the genetic code are also within the scope of the locution. Bor example, a number Of amino acids are designated by more than one triplet. Codons that speedy the same amino acid, or synonyms (for example, CAU and'CAC are synonyms for: hisbdine) may resul '1 in “silent” mutations Which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes In the amino acid sequences of the subject proteins Will exist among mammalian cells. One skilled in the art will appreciate that these variations In one or more nucleotides (up to about 3-5% of foe nueleotldes) of the nucleic acids encoding: a parttenlax protein may exist among individuals of a given species due to natural alfohc variation. Any and all such nucleotide variations and msultlng amino acid polymorphisms are within the scope of this invention.

In certain embMirnents, the mcombinant nucleic acids of the invention may be operahly linked to one. or more regulatory nncleotids sequences in an expmssion construct* Regulatory nucleotide sequences wiE generally be appropriate to the host cell used tor expression. Numerous types of appropriate expression vectors and suitable regulatory' sequences are known in the art for a variety of host cells. Typically, said ohe or more regulatory nueleotide sequences may include, but am noil imbed to, promoter sequences, leader or signal sequences, ribosomal binding sites, d&amp;oscripiiooai start and termipatiob sequences, translational start and termination sequences*: and: enhancer or activator sequences, Constitutive or induci ble promoted as known in the art ate contemplated by the invention, The promoters may be either naturally occurring promoters, or hybrid promoters that: combine elements of mom than one promoter. An expression construct may be present in a ceil on an episonte, suehas a plasmid, or the expression construct may he inserted in a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the seleetioh of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. in certain aspects of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sepuenee encoding an AetRil polypeptide and opera hly linked to at least one regulatory sequence. Regulatory sequences are art-reeognlaed and aio selected to dimet expression of the ActRIJ polypeptide. Accordingly , the term regulatory sequence includes promoters» enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddelj &amp;me Expressim Technology: Methods in Enzym&amp;logy* Academic Press, San Diego, CA (1990), For instance, any of a wide variety of expression control sequences that control the expression of a DMA sequence when operatively linked to it may be used In these vectors to express DMA sequences encoding an ActRlI polypeptide. Sueh usefhi expression control sequences, Include, for example, -the early and late promoters of SV49, tet promoter, adenovirus or cytomegaloviriis ««mediate early promoter, RS¥ promoters, the lac system, the tip system, the TAC orTRC system, T7 promoter whose expression is directed by Ύ7ΜΜΑ polymerase, the major operator and promoter regions of phage lambda , fee control regions for Id coat protein, the promo ter for 3~phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid: phosphatase, e,g., Pho5, fee promoters of the yeast «-mating factors, fee polyhedron promoter of fee baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells Or foeir viruses,, and various combinations thereof; It should foe understood that the design of the expression vector may depend on such factors as fee choice of the host cel! to be transformed and/or: the type of protein desired to be expressed. Moreover, the vector'soopy mmtber, the ability to control that copy number: and the expression of any other protein encoded by the vec tor, such as antibiotic markers, should also be considered, A recombinant nucleic acid of the invention can be produced by ligating the cloned gene, or a portionfhereof into a vector suitable for expression in either prokaryotic cells, eukaryotic ceils (yeast, avian, Insect or mammalian), or both. Expression: vehicles for .production of a mcombinant ActlUI polypeptide Include plasmids and other vectors. For instance, suitable vectors include plasmids of the types; pBB322-derived plasmids, pEMBL-derived plasmids, pEX-dersved plasmids, p8Tas--deriYed plasmids and pUC-derived plasmids fer expression in prokaryotic feeds,

Sbm&amp;tmmmuikn expression Vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic franseripbon units that are expressed: in enlaryoiie cells, ThepeBXAI/amp, peDHAlMeo, pic/GMV', pS^2gpt, pSV2tieo;pSV2-dhfr, pTk2, pRSVneo, pMSO, pSVT?, pko-neo and: pHyg derived sectors ate examples of mammalian e,\pto8Ssori vectors suitable for transfection of eukaryotic ceils. Some of these vectbrs'saie· modified with seqnenees .from bacterial plasmids, such, as pHR322, to facBitate mplicatidn and drug resistance selection in both prokaryotic and eulmiyoijg cells. Alternatively, .derivatives of viruses such as die bovine papilloma vims (BPV-1), or Epstein-Barr virus (pHEBd, pMEP-derived and p2t)5) can be used for transient expression . of proteins in eukaryotic cells. Examples of other: viral (including retroviral) expression systems can be found below in the. description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transionnatlon of host organisms am Welt known in the art. For other suitable exptession sy^temsfor both pfokatyotie and enkmyotie pells, as well as general moombibant prcxredures, see 'Molecular-X^ldmng A Labwaior? Manual, 2nd Ed., ed. by Bam brook, Friiseb and^ Mahiaris (Cold Spring Harbor laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombmaritpoiypepddes by dto use of a baculoyims expressibn system . Examples of such haeuloviras expression systems include p^L-deriyed Veetors^(such as pV1,092, pVLl 393 and pVE941), pAeUW^derived vectors (such as pActIWl), and pBlneBae-derived veetors (such as the 8-ga! containing pBlneRae 10),

In a preferred embodiment, a vector-wildbe designed lor production of the subject ActRIl polypeptides in €H0 cells, suchias a Bemv-Script vector (Stratageoe, La Jolla, Calif), ppDNA.4 vectors (Invitrogen, Carlsbad,; Calif) and pCI~neo vectors (Promega, Madison, Wise;), AS will be apparent, the subjbet gene : constructs can be used to cause expression of the subject AefRIi polypeptides in cells propagated in culture, e,g<,,io prodace proteins, including fusion; proteins dr variant proteins, for puririeafion..

This invention also pertains to a host cell transfected with a recombinant gene Including a coding sequence (e,g., SEQ ID NO: 7 of 8) for one or more of the subject ActRE polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For example, an

ActMI polypeptide of the invention. may be expressed in Imcierial cells such as £ 'CoU, Insect cells (e.g., «slog a baoui&amp;vim'eiqptie^iori.'^em}» past, or mammalian cells, Other suitable host cells are known to those skilled in the art

Accordingly, #e present invention fitrther pertains to methods of pmdnemg the •subject ActRXI polypeptides. For example, a host cell tmnsiieiedwith an expression vector encoding an AetRII polypeptide can be enlipsd under appropriate conditions to allow expressionof dte AciRli polypeptide to occur, The AetRH polypeptide may be secreted and isolated firoM a mixtnre of cells and medium containing the AeiRil polypeptide.

Alternatively, the ActEJI polypeptide may be retained eymplasmicaliy or in. a membrane fraction and the celts harvested, lysed and the protein isolated. A; cell eulfure Includes host cells, media: and other bypmduots. Suitable media for ceil culture are well known in the art. The subject AetRII polypeptides can he isolated from ceil culture medium* host cells, or both, using techniques known in the art for puttying proteins, including lornexchange ·άάί&amp;^^3^ί^ί^01 ftltradon chromatography, ultrabltration, electrophoresis, and Immurmatfimiy purification with antibodies specific for particular epi topes of the AetRII polypeptides. IP a pmferred embodiment, the ActRII polypeptide is a fusion protein containing a domain which facilitates its purification,

In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His}/entorohinase cleavage site seqnence at the &amp;dnrnnmis of the desired portion of the recombinant ActRII polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni3* metal resin. Hie purification leader sequence can then be suhsequently removed by treatment with enierofanase to provide the purified AetRil polypeptide fe g., see Hochuli et at, {1987} J. Chromaiography 411:177; and Jankneeht et at, PNAS USA 88:8972).

Techniques: ter makmgj-ibsion. genes am welltocwn, Essentially, the joining of various DMA fragments coding for different polypeptide sequences k performed in accordance with conventional techniques, employing blunfiended or stagpr-ended tennim fhr ligation, msuSction euxyme digestion to provide for appropriate terming filllngba of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation·, in another embodiment, the fusion gene can be s>mthesb-ed: by conventional techniques including automatedΙ)ΝΑ synthesisers. Alternatively, PCR. amplification of gene fiagments can be carried out using anchor primers which give rise to complemsateiy between two consecutive gene 'fragments which cun subsequently be annealed to generate a chimeric gene sequence (see, for example, Current FmtGC&amp;k m M&amp;hcuiar Biologys eds> Ausuhei et al, John Wiley A Sons: 1992). 4, Antibodies

Another aspect of the invention pertains to antibodies. Aft antibody that is specifically reactive with aft AetEi! polypeptide (e;g,s a soluble ActfUt polypeptide) and which binds competitively with the ActRif polypeptide may be used as an antagonist of AetEJI polypeptide activities. For example, by using immunogens derived fiom an ActRII polypeptide. antibodies can be made by smndard protocols (see, for example. Antibodies: A Laboratory Manual ed. by Harlow and lane (Cold Spring Harbor Press: 1988)).. A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogeniciorm of the AetRlI polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fiision protein, Tedmiques for conferring immunogefticity on a protein or peptide include conjugation to eartiem or other techniques well jmown in the art. An immunogenic portion of an ActRli polypeptide can be administered in the presence of adjuvant The progress of immunisation can be monitored by detection of antibody titers in plasma or scrum, Standard ELISA or other immunoassays can be used With the immunogen m antigen to assess the levels of antibodies

Following inmueoiaafien of on animal with an and ge n s c p repa rati on of an ActRII polypeptide, antisemcan be obtained and, if desired, polyclonal antibodies can be isolated from the serum, lb produce monoclonal antibodies, antibody-producing ceils (lymphocytes) can he harvested from an immunised animal and fused by standard somatic cell fission procedures with immortalizing ceils such as myeloma cells to yield hylmdoraa cells. Such techniques are well known in the art, and include, fee example, the hybridoma technique (originally developed by Kohler and Milstein, (l973):Hamre,:256:495-497), the human B Pelf hybridoftta Irnmunology Today, 4; 92), and the hybridoma technique to produce human monoclonal antibodies {Cole eisl, (1985) Monoclonal Antibodies and Cancer Therapy, Alau R, Llss, Inc. pp, :77-96), Hybridbrna cells can be screened immuoochemiealfy tor production of antibodies specifically reactive with an

AcfKH po!>pq>ttde aM monoclonal anbhodies isolated from a culture eontpming such hybridoma cells..

The term Antibody” as used herein is Mended ίο include fragments thereof which are also specifically reactive with a subject ActHXl polypeptide, Antibodies can be fragmented using eoitv'entional techniques and the fragments screened fox utility in the same manner as described above for Whole antibodies, For example, Ffrhfe fragments can be generated by •^seating antibody with pepsin, The resulting F{ab)2 fragment can be ireatedto reduce bisulfide bridges to produce f ab fragments, The antibody o f the present: invention is further intended to include bispeetfic, single-chain, and chimeric and himumixed molecules. having ajOBo&amp;yJb* polypeptide conferred by at least onoCDR tegion of the antibody. In piofenod embodimeuts, the antibody '.further comprises a label attached thereto and able to be detected (e,g,, the label can be n: radioisotope, fluorescent compound,enzyme ot enzyme co-factor).

In certain preferred embodiments, an antibody of the invention is a monoclonal antiha(%s and in pertain. cmbodimenis, the Invention makes available methods for generating novel antibodies. For example, a method tor generating a monoclonal antibody that binds; specifically to an AetEII polypeptide may comprise administering to a moose an amount of an humunogenic composition composing the AciEH polypeptide-effective to sfimidate a detectable immune response, obtaining andhody-ptodocing cells (e-g., cells from the spleen) from the mouse and timing the anfibody’f rodueing cells with myeloma cells to obtain and testing the antibody^producing hybridomas to identify a bybridoma that prodoeas a mcmOeolonsl antibody that binds specifically to the ActRII polypeptide. Ones obtained, a hybridoma can be propagated in a ceil calmte, optionally in culture conditions where the hybridoma-derived cells produce die monoclonal antibody that binds specifically to the AetRil polypeptide. The monoclonal antibody may be purified from the cell culture. 'The adjeetive ^specifically maefive with” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., an ActRIi polypeptide) and other antigens that are not of Interest that the antibody is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample, In certain methods employing the antibody, such, as therapeutic applications, a higher degree ..of specific ity in binding may he desirable, Monoclonal antibodies generally have; a greater .tendency· (as compared to polyclom! antibodies) to disedminaie effectively between the desired antigens and cross» reacting polypeptides. One characteristic that influences the specificity of as antibody: antigen interaction is tlto affinity of the antibody lor the antigen. Although the desired specificity may be reached wiffi a range of dif&amp;tont affinities, generally presetted antibodies null have as affinity (a dissociation constant) of about 16^.10*', ICF®, Id"9 or less.

In addition, the teehnk|nes used to screen antibodies in order to identify a desirable antibody may Influence the properties of the antibody obtained, For example, if an antibody is to be used for binding an antigen in solution, It binding. A variety of different techniques are available tor testing interaction: between antibodies and amigens: to: identify particularly desirable antibodies. Such teebnispes includeELIS As, surface plasmon resonance binding assays fe.g,, the Biacore binding assay, Ilia-core AB, Oppsaia, Sweden), sandwich assays feigi, the paramagnetic bead system of IGBN:· International, Inc,, Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohistochenhstty.

In certain aspects, the disclosure provides antibodies that hind to a soluble AeiEII polypeptide. Such sptihodies may be generated much as described above, «sing a soluble Actfill polypeptide or fragment thereof as an antigen. Antibodies of this type can he used, e.g., to detect ActRII polypeptides in biological samples arjd/or to monitor soluhie AciRII polypeptide levels in an individual. In certain cases, an antibody that specifically binds to a soluble AetMlIpoIyp^iide ean be used to htodhlate activity of au AmMt polypeptide and/or an AefcRH ligand, thereby regulating (promoting or inhibiting) growth of tissues, such as hone, cartilage, mnsclei fet, and neurons. &amp; mrnmrnMsm

In certain aspects, the present invention relates: to the ass of theaubfect AotRII polypeptides (e. g,, soluble AetEfl polypeptides) to identity compounds (agents) which, are agonist or antagonists of the AcMIl polypeptides. Compounds identified through this screening can be tested in tissues such as bone, cartilage, muscle, fat and/or neurons, to assess tlmirahil% to modulate tissue g^wthiit vitro. Optionally, these compounds can tother be tested, m ammal models to assess their ability tomodulate tissue growth in vivo.

There to* scieeiSttg^^fap^ittic agertto tor uiodnlaitog tissue growth by iargettog the ActiUI polypeptides, in certain embodiments, Ifigh-dRoughput screening of compounds can he carried out to identify agents that perhirh AetRII-mediated effects on growth of bone, cartilage, muscle, fat, and/pr neuron, In certain embodiments, the assay is carried out to; screen and identify compounds that specifically inhibit or reduce binding of an ActEII polypeptide to its 'binding partner, such as an AetRli ligand (e.g,, activto,.Nodal, GDFSy <3DFl l or BMP7f Alternatively, the;assay can be used to identify compounds that enhance binding of an AetRli polypeptide to its binding proteto such as an ActRII ligand, hr a further embodiment, the compounds can be identified by their ability to interact with an AetRli polypeptide, A variety of assay formats will suffice and, to light of the present disclosure, those not expressly described herein will nevertheless be comprehended by pne of ordinary skill in the aft . As described herein, the test compounds (agents) of toe myepdon rnpy be created by any combinatorial chemical method. Ahemaflyely, the subject compounds may be naturally Ctocurring biomotoeuies syntlmslxed to vivo or la vitro. Compounds (agents) to be tested tor their ability to act as modulators of tissue growth can he produced, tor example, by bacteria, yeast, plants or other organisms (e g., natural products), produced chemically (e.g., small molecules, including peptidomimeties), or produced mcombinaatiy. Test compounds contemplated by the presem invenftoh include sosvpeptidyl o^nie molecules, peptides, polyTteptides, peptldominteties, sugars, hormones, and nucleic acid molecules. In a specific embodiment, toe test agent is a small organic molecule having a molecular weight o Hess than about 2,000 daftons.

The test compounds of toe invention can be provided as single, discrete entities, or provided in libmries of greater complexity', such as made by combinatorial chemistry; These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of Orpnic; compounds. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially to initial screening steps. Optionally, the compounds may be optionally derivatteed with other compormds and have drmivatizing groups that facilitate isolation of the compounds, Non-limiting examples of derivatiziog groups snclnde biotin, ftooreseefe, digoxygehin, green iMoteseetti protein,, isotopes? poiyhlstidiuej magnetic beads, glutathione S riansferase (GST), pbpioaetsvatible cmsslitifeers or any combinations thereof.

Ifi many drag screening ptbgraras which test libraries of compounds and natural exhaera, high throughput assays am desirable in order to maximize the comber of compounds surveyed in a given period of time, Assays which am perforined in CelMree systems, such as may he derived with purified or semi-purified proteins, are often preferred as ‘"primary'* screens in that they can be generated to permit rapid developraePt and re iati vely easy detection of an al teration in a molecular target which is mediated by a test compound. Moraowr, the effects of ceilalar toxici^ can he assay instead being foeased primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity between an AetElI polypeptide and its binding protein (e.g., an ActRII ligand).

Merely to illtistrate, in an exemplary screening assay of IM prasent inventibn, the compound of interest is contacted with an isolated and purified AetRII pol^eptide which is ordinarily capable of binding to an AetRII Hgand, as appropriate lor the intention of the assay. To the mixture of the compound and AetRII polypeptide is then added a corappritioa containing an ActRIi ligand. Detection and quandhcarion of AetRIRAcfRIi Hgand complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating} complex formation between the ActRIi polypeptide and Its; binding protein.

The efficacy of the ^compound: eats be assessed by generstipg dose response curves from data· obtained using various cotjcentratlons of the test compound, Moreover, a control assay can also be perfomted ra ptovide a baseline .for comparison, For example, in a control assay, isolated and purified ActRIi ligand is added to a composition .containing the ActRIi polypeptide, and the fbnnaiion of ActRIFAotRIl ligand complex, is quantitated in the absence of the test compound, It will be understood that, in general* the order is which: the reactants: may be admixed can be varied, and can be admixed simultaneously. Moreover, in place of purified pmteins, cellular extracts and lysates may he used id tender a suitable cell-free assay system.

Complex fermailpn between tire ActRIi polypeptide and its binding protein may be detected by a varie ty of teehniqnes. For instance, modulation of the formation of complexes can he guaniitated using, lor examplsi delectably labeled proteins such as radiolabeled (e,g·* iSS, >4C ot:*H), fitsoreseenity :&amp;he!ed fe.g,, FTTC), orenxymafieally labeled AetKlI polypeptide or its binding protein, by immunoassay, or by chromatographic detection.

In certain embodiments, tbs present invention contemplates the ass of llnoresceaee polanzation assays and: lluoreseenee resonance energy transfer (FRET) assays in measuring, either directly or intemefipin' between an ActRfi polypeptide and its binding protein. Further, other modes of detection, such as those based oo optical waveguides (PCTPublication WD 96126432 and U.S. Pat, No, 5,622,196), surface plasmon resonance (BBR), surfacecharge sensors, and surface force sensors, are compatible with mar^ embodi ments of the invention.

Moreover, the present invention contemplates the use of an interaction imp assay, also known as the “two hybrid assay,*" for identifying agents that disrapt or potentiate inteiaction between an ActEII poly^ptide and its binding protein, See for example, U.S, Pat. Flo. 5,283^17; Esrvos et at. <5 993} Cell 72:223-232; Madura et ai. (1993} I Biol Chem 268; 12046-120S4t Bartel et at. (1993} Biofeehnigises 14:920#24j and iwabechi et al. (1993} Oncogene S'1693-1696), In a specific embodiment, the present invention^Contemplates the use of reverse two hybrid systems to idehtsfy compounds (e ,g., small molecules or peptides} that dissociate interactions between an: ActlMI polypeptide and its binding protein. Sec for example, Vidal and Legrain, (1999) Btteleie &amp;e$klU>8'27t919-2^γί^ί:βΐ^ί^$χ^ΐϊϊ, (1999) Trends Biotecbnoi 17;374~81; and U.S. Pat Bos. 3,525,490; 5,955,280; and 5,965,368. lit certain embodiments, the subject compounds are identified' by their ability to interact wstlt an ActRlI polypeptide of the invention. The iniemetfoh between the compound and tire ActEII polypeptide may be covalent: or nod-oova!eni For example, soph interaction Cap be identified at the protein level using in vitro biochemical methods,including photo-crossltnking, radiolabeled ligand binding, and affinity chromatography (Jskoby WB et al, ! 974, Methods in Brteymplogy 46:1). In pertain cases, the compounds may be screened: in a meebanism based assay, Snefe as an assay to defect compounds which bind fo an AetMl polypeptide. Ibis may include a solid phase or fluid phase binding event. Alternatively, the gene encoding an : ActEH polypcptile can be transfected with a reporter system (e,g.,:p;~ galaciosidase, lusiforase, or green fluorescent protein): into a cell and screened against the libraryipreferahly by a high throughput screening or with individual members of the library. Other mechanism based binding assays may be used, fat example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized aptibody· or resolved by capillary electrophoresis, The bound compounds may be detected usually using colorimetric or Suomsceaee or surface plasmon msonanee. la certaih aspeciSj, the present invention provides methods and agents for sbmukdug muscle growth and increasing muscle mass, for example* by antegomfong hinctlons of an AciE.II peiypeptkle and/or an ActRli Ujgshfo- Therefore, day compound id&amp;atiited can be tested in whole cells or tissues, in vitro or id vivo, to confirm their ability to modtiiate rnnscle growth, Various methods known m foe art can be Ufotod fof this purpose. Far example, mefoods of the invention are performed such that foe signal transduction through an AetRIl protein activated by binding to an AetRli ligand (e.g., GOFd) has been reduced or inhibited.

It will be recogstiaed that foe growth of musc le tissue i n foe organism would result in an increased muscle mass in foe organism as compared to fee muscle mass of a corresponding organism (or population of orgarnsms) in which the signal transduction through an ActRil protein had not been so effected.

For example, foeeffeeipf foe ActK|i|folypefoidea or test compounds onhmscle cell groWtlFpfoii fetation can he ^tenhiUed by measuring gene expression of ?Ρχ-3 aM Myf-S which are associated with proliferation of myogemc cells, and gene expresskmof MyoD which is associated with muscle differentiation£©,g.5 Amfoor et at, Dev Biol, 2002,251:241-57), It is: known that GDF8 down-regulates gene expression of Pax-3 and Myi-$, and pre vents gene .expression of MyoD. the AetBlI polypeptides or test compounds am expected to antagonize this activity of G0F8, Another example of call-based assays includes measuring the proliferation of myoblasts such as €(2}€(12) myoblasts in the presence of the ActSIf polypeptides or test compounds (e,g., Thomas et at, J Biol Chem, 2000,275:40235-

The present Invention also Confemplates in viyo assays to measure muscle mass and strength. For example, Whittemore et al. (Blochem Bibphys Bes Common, 3005,300:965-71) discloses a method of measuring increased skeletal muscle mass and increased grip strength: in mice. Optionally, fotsmefoodean be used fo defermiae therapeutic effects of test compounds (e,g., Actlill polypeptides) on muscle diseases orconditions, for example those diseases. Cor which muscle mass is lira king;

In certain aspects, the presentin' vendors provides methods and agents lor modulating (stimulating or irfeihiting) boss formation and increasing bone mass, Therefhre, any compound identified can be tested in whole cells or tissues, in vitro or m vivo, to confmo their ability to modulate bone or cartilage powth, Various nieihods known m fee art can be utilized for this purpose.

For example, the effect of the Aet.R.0 polypeptides or test compounds on bone or cartilage growth can be determined by measuring induction of Msx2 or differentiation of osteoprogeniior cells into osteoblasts in cell based assays (see, e,g., fbtiuiski et al., Nat Genet. 2001, 27(1):|4~S; pine et al,, Front Biosei. 2Q0% 9- 1520-9). Another example of cell-based assays inelpdei analyzing the osteogenic aetiGty of fee snbioet AefEil polypeptides and test compounds in mesenebyma! progenitor and osteoblastic cells, fee illustrate, recombinant adenovinrses expressing an AciRII polypeptide wereinstructed to infeet pluripotent mesenchymal progenitor OHlOTi/2 ¢¢11¾ predsteoblastic C2CI2 cells, and osteoblastic TB-85 colls. Osteogenic activity is then determined by measuring fee induction of alkaline phosphatase* osteocalcin, and matrix mineralisation (see, e.g.« Cheng et ai,Jhsne Joint Surg; Am. 2003:, 85-A(8):l 544-52).

The present inyentipn also contemplates in vivo. growth. For example,Matnkuo,g-Mattbai etal, Bone, 28:80-85 (2001) discloses a mt osteoporotic mode! in which bone repair during the early period after fracture is studied,.

Kobo et ai.s Steroid Biochemistry &amp; Molecular Biology, 58:197-202 (1999) also discloses a rat osteoporotic model in which bone repair during fee late period after fracture is studied. These references are incorporated byreferenceherein in their entirety for their disclosure of rat model for study on osteoporotic bone iracture, In certain aspects, the present invention makes use of tmetem healing assays feat ate knotsm in the art. These assays include fracture technique, histological analysis, and biomechanical analysis, which are described in, for example, fJ.S. Pah No. 6,521,750, which is incorporated by reference in its entirety for its disclosure Of experimental protocols fer causing as well as measuring feeextent of fractures, and fee repair process.

In certain aspects, the present invention provides methods and agents for controlling weight pin and obesity. At fee cellular leys!, adipocyte proliferation and differentiation Is critical in the development of obesity, which leads to the pnemtion of additional fat cells (adipocytes). Therefore, any compound identified can he tested hi whole cells or tissues, in vitro or is vivo, to confirm their ability fo modulate adipogenesis by rneasming adipocyte ptoMfeaiion or diffemstiaaaa. V'atioos methods known in the artennhe «dimed fbrthte purpose. For example, the effect of an AetRII polypeptide fo.g,, a soluble ActRII polypeptide) or mst eompormds on adipopaesis cau be determined by measuring difTereniiafion of 3T3-LI prePipeoytes to mature adipocytes in eel! based assays, such as, by observing the accumulation of tnscylgiyeerel in Oil Red O staining vesicles and by the appearancebfcerfamadlpoeyte marfeers#cb as FABP (aF2M22) and PFARyl, See, for example, Rsuseb et ab, 2000, Mol Ceil Biol, 20:1008-¾ Deng pi a!., 2000, BndoeSnology, I4f :2370-01:1011:0101:.,:2000, fohes lies. 8:240-54. Another exampleofeell-based assays includes analyzing the role of ActRII polypeptides and test compounds In proliferation of adipocytes or adipocyte precursor cells (e.g., 3T34J cells), such as, by nionitoring bfomodeaxyuridine CBrdU)-po$itive ceils. See, for example., Rico et aL> 1998, Mol Cell iioehem. 189: 1-7; Masuno et a!., 2003, Tended! $ck 25:314-20, it is understood that foe screening assays of the pfoseot invention apply to not only the: spbiect AetRlI polypeptides and variants of die ActRII polypeptides, but also any test compounds including agonists and antagonist of the ActRII polypeptides. Further, these screening assays are usefo! for drag target ven&amp;adon and quality control purposes, % Ex.emp|a!ty,;ihetiaife.llsgs

In certain emhodi menis, composMens (e,g,5 ActRII polypeptides) of the present invention can be used for treating or preventing a disease or condition that is associated with abnonnal acti vity ofan ActiUl polypeptide and/or an ActRII ligand (e.g,, GDF8). These diseases, disorders or conditions are generally referred to herein as “ActRII-associated conditionsA In eettais emlfodiments, the present invention provides me thods of treating or preventing an individual in need thereof through administering to the individual a therapeutically elieetive amount of an ActRII polypeptide as described above. These methods are pariicnlariy aimed at therapeutic and pmphylacttefp^'^«tets::df animals, and more particularly, humans,

As used herein, a therapeutic foat “prevents” a disorder or condition refers to a compound that, In a statistical sample, reduces the occuncnce of dm disorder or condition In the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of o»e or more symptoms of the disorder or condition relative to the untreated eordrol sample. The term "treating”' as used herein includes prophylaxis of the oanjed condition or amelioration or elimination of the condition onse it has been established.

AetRII/ActRIIligand complexes play essential roles in tissue growth as well as early developmental processes such as the correct formation of various -structures or in one or more post-developmental capacities including sexual development, pituitary hormone production^ and creation of bode and cartilage. Thus, AhtRIBasseeiated conditions include abnormal tissue growth and developmental defects. Ih addition, ActRil-associated conditions include, but are not limited to, disorders of eeiVgrowth and diffetbotiatiou such as inflammation, allergy, autoimmune diseases, infectious diseases, and tumors.

Exemplar AmtRli'associated conditions include neuromuscular disorders (e.g., muscular dystrophy and muscle atrophy), congestive ohstruch ve pulmonaty disease, muscle wasting syndmme, sateopeniai cachexia, adipose tissue disorders (e.g., obesity), type 2 diabetes, and bone degenerative disease (C.g., osteoporosis). Other exemplary AetRli-associated conditions include muscidodegeneratwe and neuromuscular disorders, tissue repair (e g., wound healing), neurodegenerative diseases (e.g., antyotrophie lateral Sclerosis), immunologic disorders (e.g., disorders misted, to abnormal proliferation or fnnedoa of lymphocytes), and obesity or disorders related to abnormal proliferation of adipocytes.

Is certain embodiments, compositions (e.g>, soluble Act!.).! polypeptides) of the itweniion are used as part of a treatment for a muscular dystrophy . The term "muscular dystrophy’' refers to a group of degenerative muscle diseases characterized by gradual w'eahehing and deterioration of skeletal muscles and sometimes the heart and respiratory muscles. Muscular dystrophies are genetic disorders characterised by progressi ve muscle wastiog and weakness feat begin with microscopic changes in the muscle. Asmusefes degeherafe over timUi the person’s muscle strengih declines. Exemplary muscular dystrophies that-can be treated with a regimen including the subject AetRII polypeptides include; Buehetme Muscular Dystrophy (BMD), Becker Museuiar Dystrophy (BMP), Empry-Brelfuss Muscular Dystrophy (B.DMD), Llmh^Girdle Muscular Dystrophy :F^teso^e:iohb^^l;Muscular Dystrophy|FSH or f$HD) (also known as lApdoWsy-Dejerine), Myotonic Dystrophy (MMD) (also known as Steinerfs Disease), Oculopharyngeal Muscular Dystrophy (QEMD), Distal Museuiar Dystrophy (DP), Congenital Muscular Dystrophy (CMP)·

Duehenne Muscular Dystrophy (DMD) was first described Ijy fire French neurologist Guillaume Bsajamiu AmaM Duchenae In the 1 S6f>a. Becker Muscular Dystrophy (BMD) is named after the German doctor Peter Emil Becker, who first described this variant of DMD in the !950$, BMP is one of the mosiireqnent inherited diseases k males, affecting one in 3 ,500 hoys. DMD oecum when the dystrophin gene, located on the short ana of the X chromosome, is broken, Since males only carry one copy o f the X chromosome, they only have one copy of the dystrophin gene. Without the dystrophin protein, muscle is easily damaged during cycles oiedniraction and relaxation. While early in the disease muscle compensates by rsgenemtioh, later on muscle progenitor cells eanuot keep up with the ongoing damage and healthy muscle Is replaced by non-functional flbro-fatty tissue,: BMD results from different mutatious k fee dystrophin gene, BMD patients have some dystrophin, but it is either insufficient in quantity or poor in quality, Hakng some ;'^h^^ip\pi0ieci$''^m:'n^se!es of those with BMD front degenerating as badly ores quickly as those of people with DMD.

For example, recent resist Mocking or eliminating function df 0DF&amp; (an AefRH ligand) in vivo can effectively treat at least certain symptoms in DMD and. BMD patients (Bogdanovich: et al,, supra; Wagner et ah, supra), Thns, the subject Actfell polypeptides mayset as DBFS inhibitors (antagonists), and constitute an alternative means of blocking the ftmetions: of ODFh andkv ActRff in vivo in DMD and BMD patients.: .Similarly, the subject ActBII polypeptides provide an effective means to increase muscle mass in other disease conditions that are in need of muscle growth. For example, GonMlea-CadaVld et aL (supra) reported that that CDF8 expression eomdatesdnvetsely with £atefree mass in humans md that increased expression of the 0DF8 gene is associated with weight loss in men With AIDS wasting syndrome,: By inhibitkgihe mnctibh dfGDFS in AIDS patients, at least certain synnptomsbiMDS may be- alleviated, if not completely eliminated, thus significantly improving quality of life in AIDS patients.

Since loss ofGDFfifea Aetlili ligand) function Is also associated With fci loss withoni diminution of mttrient intake (Zimmers et ah, supra; McPherrop and Lee, supra), the subject ActRIi polj'peptides may further be used as a therapeutic agent for slowing· or preventing the development of obesity and type If diabetes.

The .eaftcer anorexia-cachexia syndrome debilitating s«d life- fereatening aspects of cancer,: .Ρί^^^Ι^Λνβΐί|&amp;ΐ'Ι®8ΐ':ίϊ):^ίιι«^ anoj^xia-caehexia ^«drome is a common feature of many types ofeancer and is responsible net only for a poor quality of hie: and poor response to chemotherapy, but also a shorter survival time than is found In patients wife comparable tumors without weight loss. Associated with anorexia, fat and musciefissue wasting, psychological distress, and a lo wer quality of life, cachexia arises "from a complex interaction between the cancer and the host, ft is one of the most common causes of death: among cancer patients and is present Ip Sp% at de&amp;fe, ft is a complex example of metabolic chaos effecting protein, carbohydrate, audjat metabolism.. Tumors produce both: direct and: Indimet abnormalities, resulting in anorexia and weight loss. Currently, there is no treatment to control or reverse the process. Qspapf· airotoxia-caclrexia syndrome affects cyfokinO produefion, release of lipid-niobillzing and profeOlysisAnducing factors, and alterations in intermediary metabolism- Although anorexia Is common, a decreased food intake alone&amp;umblefo,aoemmt for the changes in body composition seen in cancer patients, and inereasitigmntrient intake, is unable: to reverse: the wasting syndrome. Cachexia should be suspected in patients wife cancer if an: involuntary weight loss of greater than five pereentoi premerbid weight occurs within a six-month period.

Since sysfemin overexpression of ClpfiS in adult mice was found to induce profound muscle and fet loss analogous to feat seep in human cachexia syndromes (Ziouners et al, supra), the,subject:Ac^f''polyp^ttdea as pharmaceutical compositions can he beneficially used to prevent, beat, or alleviate the symptoms of fee cachexia syndrome, where muscle growth is desired,

In other eofbodinmntx, the present invention provides methods of inducing hone and/or cartilage formation, preventing hone loss, increasing hone mineralization or preventing the demineralization ofbone, For example, the subject ActiOI polypeptides and compounds identified ip the present invention have application in treating osteopofosis and the healing of bone fmetures nod cartilage defects in humans and other animals, ActRIl polypeptides may be nsefoi in patients that are diagnosed with subciinieal low bone density, as a protective measure against fee development of osteoporosis. i n one specific embodiment, methods and compositions of the present invention may-' find medical uti lity i n the hea ling of bone fractures and cartilage defec ts in humans a nd other animals. The subject methods and compositions may also have prophylactic use in closed as well as open fracture mducbon and also in fh^dmproved fixation of artificial joints- $e mvp bone formation induced by an osteogenic agent contributes to the repair of congenital, baumaTndused, or oncologic resectioninduced craniofacial defects* and also is useful in cosmetic plastic surgery. Further, methods and compositions of the invention may be used in the treatment of periodontal disease, and in other toodr repair processes. In certain cases, the subject AetM polypeptides may provide an environment to attract honeyfbmuog cells, stimulate growth of bone-forming eelfe or indtme diiforemimion Of progenitors of bene* fornting cells. Aetfell polypeptides of the invention may also be useful in the treatment of osteoporosis, Further, ActRIf polypeptides may be used in oatiilage delect repair and pmveniion/reversal Of osteoarthritis.

In another specific embodiment, the invention provides a therapeutic method and composition for repairing fractures and other conditions related to cartilage and/or bone detects or periodontal diseases. The invention further provides therapeutic methods and compositions for vvoond healing and tissisempair. The types of wounds include, hut are not limited to, bums, incisions and ulcers. See e.g., PCTBsblication Flo: WOS4/01106: Such Compositions comprise a therapeutically effective amount of at least one of the AcfRII polypeptides of the inyention In admixture with a pharmaceutical iy acceptable vehicle, carrier opmatrix.

In'another specific embodiment, methods and compositions of the invention can be applied to conditions causing bone loss snob as osteoporosis, hyperparaibyroidism, Cushingfe disease, thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubular acidosis, Or anorexia nervosa. Many people fenow that being female, having a: low body weight, and leading a sedentary lifestyle are- tisfc .(Ihiss Of bone m ineral density, leading to fracture: risk). However, osteoporosis cats also result feoni the long-term use Of cerfein medications, Osteoporosis resulting from drugs or another medical condition is known as secondary osteoporosis, In a condition: .knows as Cushing’s disease, the excess amount of cortisol produced by the body results in osteoporosis and fractures. The most common medicaiionS bssociated with secondary: OsfeoporcsiS: bfe the eofficosteroids, aelass: of drugs that act like cortisol, a hormone produced ..naturally by the adrenal glands, Although adequate levels of thyroid hormones (which are produced by the thyroid gland): are: needed tor the development of the skeleton, excess thyroid hormone can decrease hone mass over time. Antacids that eoniaihalnmimwn can lead fe bone loss when taken: in high doses by people wrih kidney problems, parhculady those undergoing dialysis. Other medications that can cause secondary osteoporosis include phenytoin {Dilantin} and barbiturates that are used to prevent seizures; mefeotrexste (Rheumatrex» Immimex, Folex PPS)» a drag for some forms of arthritis» cancer, and immune disorient; cyelosporlne (Sandimmune» Neoral), a drag used to treat some autoimmune diseases and to suppress the immune system in organ transplant paifems; luteinizing hormone-releasing hormone agonists (Lepron, Zoiadcx). nssd to treat prostate cancer and endometriosis; heparin {Caielparme, Ii§«aemin), an anticlotiing medication; and ehoiestpamme (Questran) and colestipol ^Golestid), used to treat high eholesterol Gum disease causes bone loss in our mouths force our bodies to defend against them. The bacteria poadnce toxins and enzymes under the gum-line, causing a chronic infection. in a further embodiment» the ptesent^mv€ptip9'^>^s^^c^::aρd^^»p¢μ'ttc agents for treating diseases or disorders associated with abnormal or unwanted bone growth. For example» patients having the (FOP) grow an abnormal "Second sheleton” that pmvents any movement. Additionally» abnormal bone growth can occur after hip replacement surgery and thus ruin fee surgical on tcorae, This is a more common example of pathological hone growth and a situation in which the subject methods and compostlions may he ihempsotioal ty useful. The same methods and: compostlions may also be useful for treating other forms of abnormal bone: growth (e»g,» pathological growth of hone following trauma, bums or spinal cord injury), and for treating or preventing the undesirable conditions associated With the abnormal bone growth seen in connection with metastatic prostate cancer or osteosarcoma. Examples of these: therapeutic agents include, but ate hot limited ito, ActRII polypeptides that antagonize function of as ActRII ligand (e.g., BMP?)» compounds that disrupt interaction between am ActRII and its ligand (e.g,, BMP?), and antibodies feat:specifically bind to an ActRII receptor such that an ActRfi ligand (e.g,, BMP?) eannpt bind to the AetR.O receptor.

In other embodiments, the present invention provides compositions and methods: for regulating body fat content in an anintal and for treating or preventing conditions related thereto, and par%uiafly, health-compromising conditions related thereto. According to the present invention, te mplate (control) body weight can refer to reducing or increasing body weight, reducing or-increasing the ram of weight gain, or increasing or rednemg the rate of weight loss, and afeo mchtdes actively maintainiug, or not significantly changing body weight fe,g., sgais^st extenmi or mlcrnsl influences Ulrich may otherwise increase or decrease tody weight), One embodiment of the present invention relates to regulating tody «eight by adminismring to an. animal (e.g., a human) in one speeitic emfeodirnentj the presem invention relates to methods and compounds lor reducing body weight and/or reducing weight gain in asaoimaL add: more pmtierdarly, for treating or ameliorating obesity in patients at risk for or suffering from obesity, In another specific emtodirneni, the present invention is directed to methods and compounds for treating an animal that is unable to gain or retain weight (e.g;,, an animal with a wasting symdrome), Snell methods are effective: to increase body weight and/or mass , or to: reduce weight and/or mm loss, or to improve conditions associated with or caused by undesirably tow (e.g., unhealthy) body weight and/or mass.

In other embodiments, the: subject AetRIi polypeptides can be used to form; pharmaceutical compositions;.fh^t/can be benetieialiy used to prevent, treat, or alleviate symptoms of a host of diseases involving nemodogeneratiom While not wishingto he toned by any .particular ttootyj the .subject ActRIl polypeptides nay antagonize the inhibitory feedback mechanism mediated through the typo 1 receptor ALK|, thus allowing new neuronal growth and differentiation. The subject AetMI polypeptides as pharmaceutical compositions can be beneficially nsed to prevent, treat, oraMeviafe symptoms of diseases with neurodegeneration, ineluding Alzheimer’s disease tAO), ParkinsonfeTJisease (P0), Amyotrophic Lateral Sclerosis (ALS), and Ifonfingfonfe disease (HD)* AD is a chronic, incurable, and &amp;έίί^δ^ nervous sysfem XCNS) disorder that occurs gradually, resulting in: memory loss, unnsuai behavior, pemonality ehatiges. and: a decline in thinking abilities. These losses are related to the death of specific types of brain cells and. the breakdown of connections between them- AD has been described as childhood. - dewlopment in reveme. In most people with AD. symptoms appear after the age 60. The earliest symptoms iuehide toss of recent memory, itolfo judgment, and changes in personality. later in the disease, those with AD may forget tow to do simple tasks like 'Washing their hands. E ventually people with AD lose all reasoning abilities and become dependent on other people for their everyday care. Finally, the disease becomes so debiStaiing drat patients are hedrtdden and typically develop coexisting illnesses, AD patients most eonunonly die fton pneumonia, S to 29 years from disease onset. FD i s a chronic, incurable, and unstoppable CHS disorder that occurs gradually and results in uncontrolled body movements, rigidity, tremor* and gait difficulties. These motor system problems are related to the death of hmin cells inan area of the brain that produces dopamine, a chemical that helps control muscle activity. In most people with PD, symptoms appear after age SO. The initial syrnptonis of F D as» a pronounced tremor affecting the extremities, notably In the hands or lips,Subsequent eharaeteristio ayrnptoms o f FD are stillness or slowness of movement, a shuffling walk, stooped posture, arid impaired balance. There are wide ranging secondary symptoms such as memory loss, dementia, depression, emotional changes,; swallowing difficulties, abnormal speech, sexual dysfunction, and bladder and bowel problems. These symptoms will begin to interfere with routine activities, such as holding a fork or reading a newspaper. Finally, people with PD become so profoundly disabled that they am bedridden. People with FD usually die from pneumonia. ALSj also: called Lon: Dehrig’s disease (motor neuron, disease) is a chronic, incurable, and unstoppable ¢3148 disorder that attacks the motor ppumns, components of the DHS that connect the brain to the skeletal muscles, in ALS, Ihemotor neurons deteriorate and eventually die, and though a person’s brainnormally remama fully functioning axicl alertrihe command to move never reaches the rhascles. Most people who get ATS are between 40 and f0 years old, The Srst motor neurons that weaken are those leading to the arms Or legs.

Those with ALS may have trouble walking, they may drop things, fall, slur their speech, and laugh or cry uncontrollably. Eventually the muscles In the limbs begin to .atrophy from disuse. This muscle weakness will hecome debilitating and a person Willneed a Wheel chair or become enable to function out of bed. Most ALS patients die from respiratory failure or from eontplicaioin! of ventilator assistance like pneumonia, 3-5 years from disease onset.

The Pauses of these neurological diseases have remained largely unknown. They are conventionally defined as distinct diseases, yet clearly show extraordinary similarities in basic processes and commonly demonstrate overlapping symptoms far greater than, would be expected by chance aldne,: Cppent disease dehnitions fell to properly deal with the: issue of overlap and a new classification of the hcnrodegenerahve disorders has been called fhr. HD is another neurodegenerative disease resulting from geneticaUy programmed degeneration of neurons in certain areas of the brain. Ibis degeneration causes uncontrolled movements, loss of intelteetuai faculties, and emotional disturbance, HD is a familial disease, passed from parent to child through a dominant mutation in the wild-type gene. $»me early 'symptoms of HD 3½ mood swings depjcsriotg irritability or trouble driving, learning new things, temendsering a fact, or making a decision. As the disease progresses, increasingly difficult and the patient may have difficulty feeding himself or herself and swallowing. The rate of disease progression arid the age of onset vary from person to person,

Tay*Sacfes disease and Sandhoff disease are glycoltpid storage diseases caused fey fee lack of lysosoiriai pdwKOsaminidase (Gravel etal., in The Metabolic Basis of inherited Disease, eds. Seriyeret at, McGraw-Hill, New York, pp 2839-2879,1995). In both disorders, GM2 gaoglioside and related glyeolipid substrates for (i-hexosaminidase accumulate in the nervous system and trigger acute neurodegeneratiors, in the most se vere forips, the onset of symptoms begins in early mfimcy, A precipitous nenrodegenerative c€at^g:':9ip..ep^4>. exhibiting motor dysfunction, seizure, visual loss, and deafness, Death usually oceans by 2*5 years of age. Neuronal loss through an apoptotic mechanism has been demonstmied (Huang et at., Hum. Mol. Genet. .6:1879-1885,1997).

It is well known that apoptosis plays a role in AIDS pathogenesis in the immune system. However, HlV-l also induces neurological disease. Shi et al. (J. Clin, Invest. 98: 1979*1990, i99b) examined apoptosis induced byHFWI infection of fhecentral nervous System (CNS) ih an in vitro model and in braid tissue from AIDS patients, and;;found feat HfV-i infectioh of prifeary feriiin culturesihdneed apoptosis in neurons and astecyfeam vifeo, Apoptosis of aeuffihs ahcl astrocyfesWasiIso defected in brain tissue from Iffif 1 AIDS: patients, iseiudipg 5/5 patients wife HIV4 demeutiantid 4/5 nondemented patfents.

Neurona l loss is also a client feature of prion diseases, such as Cre»^lbidt~Jakob disease in human, BSE ineahlerinad cow disease), Semple Disease in sheep and goats, and feline spongiform encephalopathy (FSE) In cats.

The subject ActRii polypeptides are also useful to prevent, treat, and alleviate symptoms of various PNS disorders, such as fee ones described below. The PNS is composed of fee nerves feat lead to or branch; off horn fee CN S. The peripheral nerves handle a diverse array of functions in fee body, including sensory, motor, and autonomic functions. When an individual has a peripheral neuropathy, nerves of fee DNS have been damaged. Nerve damage can arise from a number of causes, such as disease, physical iniury, poisoning, or malnutritiom These agents may affect either afferent or efferent nerves.

Depending on the cause of damage,, thpnerve sell axon, its protective myelin sheath, or both may be injured or destroyed.

The' term “peripheral neuropathy” encompasses a wide range of disorders id! whichthe nerves outside of the: brain and 'spinal cord~~-peripfseral nerves-™-have bean damaged. Peripheral neuropathy may also be referred to as peripheral neuritis, or if many nerves are involved, the terms polyneuropathy or polyneuritis may he used.

Peripheral neuropathy is a widespread disorder, and them are many underlying causes. Some of these causes are common, such as diabetes, and others are extrtenely rate, such as aptylamide poisoning and certain inherited disorders. The most common worldwide cause of peripheral neuropathy is leprosy. Leprosy is caused by the bacterium Mycobacterium leprae, which attacks the peripheral nerves of affected people, According to statistics gathered by the World Healilt Organfeation, an estimated 1.15 million people have-leprosy worldwide.

Leprosy· is extremely rare imtheiTlnited States, where diabetes is the most commonly known cause of peripheral neuropathy, it has been estimated that more than 1? million people In the Edited States and Europe have diabetes-related pofyneutopaShy, Many henropathles arc idiopathic ~ no known causncartbe found. The most common of the inherited peripheral neuropathies in dm Edited States is Charcdt-Marie-Tooth disease, Which affects approximately 125,bbb persons,

Another of the better known peripheral neuropathies is Gui!tein~Band sjmdrome, which arises from complications associated with viral illnesses, such as cytomegalovirus, Epstoin-Barr virus, and human immunodeficiency vims (BTV), or bacteria! infection, including Campylobacter jejuni and Lyme disease;. The worldwide ineidence rate is approximately 3 ,7 cases per ΪΟΟ,ΘΟΟ people annually; Other well-known causes of peripheral neuropathies include chronic alcoholism, infection of the varieella-aoster vims, botelisrn, and poliomyelitis, Peripheral neuropathy may develop as a primary symptom, Or it may be; dad to another disease. For example, peripheral neuropathy is only one symptom of diseases such as amyloid neuropathy, certam oaucers, or inherited heumlogic disorders. Such diseases may aliecf the peripheral nervous system (PNS) and the central nervous system ;(CNS), as well as other body tissues. vritbdfie subject ActRff polypeptides Include: Brachial Plexus Menrr^athies {diseases of the cervical and Srst thoracic roots, nerve tmnfcs, cords, and peripheral rtorve components of the brachial plexus. Clinical manifestations include regional paid, paresthesia; muscle weakness, and decreased sensation, in the upper extremity These disorder may be associated with trauma, iseltt^¾g·¾Mi½jUtίesp^prado.#tttlet^?yή^«Dm€ζ neoplasms, neuritis, mdiodtetapy; and other conditions. See Adams nt ai^ Ttiueiples of Neurology, 6th ed, pppSl-2}; XJiahefic Neuropathies (peripheral, aufeuotnic, and cranial nerve disorders thatore associated with diabetes nielhtus). These conditions usually result from diabetic mi^vascd|j^dn3:^'fe.voIy^ small blood vessels that supply nerves (yasa nervorum): Relatively common conditions which may be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; moooneeropahry mnlfipfex; diabetic amyotrophy; a painfel polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy {see Adams et ah, Prioesples of Meurologyy 6th ed, pl325); mononeuropathies {disease or Ranma involving a single peripheral nerve in isolation, or out of proportion to evidence of diffuse peripheral nerve dys&amp;nction), Monortemopafhy multiplex refers toa condition ehamcferiKed hy multiple isolated nerve injuries, Monofrearopatbies may result from a wide yariciy of causes, including ischemia; traumatic injury; compression; connective tissue diseases; eatoulatiye ffeumu disorders; and other conditions); Neuraigia (intense or aching pain that occurs along fee course or distribution of a peripheral or cranial nerve}; Peripheral Nervous Systom Neoplasms {neoplasms which arise from peripheral nerve tissue. This includes neurofifernmas; Schwannomas; granular cel! tumors; and malignant peripheral nerve sheath tumors, See DeVito Jr et aL, Cancer: Principles and FrautiUe of Oncology, ,6th ed, ppl 760-1}; Nurve Cornpression Syndromes (mechanical compression pf nerves or nerve roots from internal or external causes, These may result in a cohductldn blpch to horvu impulses» due to, fer example, myelin sheath dysfunction, or axonal loss. The nerve and nerve sheath injuries may be caused by ischemia; mflammsilon; a direct mechanical effect; or Neuritis (a general term, indicating inflammation of a peripheral or cranial nerve). Clinical manifestation may include pain; paresthesias; paresis; or hyperthesia; Polyneuropathies (diseases: of multiple peripheral oerves), The various forms are categorised by the type of nerve affected {e,g;, sensory, motor, or autonomic}, by the distribution of nerve injury (mg., distal vs, proximal}, by nerve component primarily affected (e.g,, demyebnaling vs, axonal), by etiology, or hy patiern of inheritance. 7, Pharmaceutical .Compositions in eermimemhodiments, compounds |e..g>., ActRII' polypeptides) of the present invention are formulated with a phannacenncaily acceptable canicr. For example, an ActRII polypeptide can fee /administered alone or as &amp; cpmpostent of a pharmaceutical formulation i&amp;crapeuhc composition), The subject compounds may be fosmttlated 1½ admipisteiioo k any convenient way % css in human or vemrirntry medicine. 1« method of the laveotiod tnctodes admimslerlngthe composition topkaily, systemically, or locally as an implant or device. Wlmd administered, the ''i^emj^n^drcoMpgtn'tiion fob use in fois invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably bo encapsulated or mjeckdfk^va^nslor^ifdr.^liv^y to a target tissue slk fe.g., fepne, cartilage, muscle, fat or neuron), for example, a sire having a tissue damage. Topical administtafion may be smtabfofor wound healing and tissue repair- Therapeutically useful agents other than the ActRII polypeptides which pray also optionallybeincluded in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with:the sukectcompounds: (e>g., ActRII polypeptides) in the methods of the invention, in certain enfoodiments, compesMbnsI of the present inydnlion may include a matrix capablakf delivering:; Oho or more: kerapeutic compounds {e.g,,:Ab®B polypekfoes) to a target tissue site{e.g,, bone, cartilage,muscle, fat or neuron.), providing a structure for the developing tissue and optimally capable of being resorbed: Into the body. For example, the matrix may provide slow release of the ActRII polypeptides. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on hiocompatibiiity, biodegradability, mechanical poperties, cosmetic appearance and hfterface properties, The particular application of the subject compositions vvlll define the appropriate ibtmulation. Potential matrices for the compositions: may be biodegradable and Chemicaily defined calcium sulfate, tnealciumphosphate, hydroxyapatite,poiylactic acid and polyanhydrides, Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proiems or extracellular matrix components. Other pdkbilal matrices are such as simemd or other ceramics. Matrices may bo composed of comMoatiohs of any of dm above mentioned types of materlai, such as polyiactic acid and hydroxyapatite or collagen anti £rioaIeintn.pk>spfeat«, Ί3&amp; bioceramics may 'ls©.;*Iter€^5 a» composition, such as in caiciuni-atepmate-phosphato and processing to alter pose siae, particle size, particle shape; and blodegradability, in certain emlKKliroents,'meihe$S:-bfiftm invention can be administered for orally, e.g.., in the form of capsules, cachets, pills, Mfilets, lozenges (using a flavored basis, usually sucrose and acacia or tragaeanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, of as lipoid 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 tlw like, each cordaining a predetanined amount of an agent as m active ingredient. An agetn may also be administered as a bkus, eieemayy or in solid dosage ferns for oral administration (eap^rles, tablets, pills, dragees, powders, granules, and thedike),: one or more tberapeune coropoands of the present invention may be mixed with pbe dr mote pharmaceutically acceptable carriers, such as xt>dinm bitrate Or dkalcmm phosphate, and/or any of the ipnowing: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example*; ca^oxymethyloellntbse, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; ¢3} hurnectants, snch as glycerol; ¢/1) disintegrating agents, such as agar-agar, ealorum carbonate, potato or tapioca starch, afginie acid, eeidatn silicates, and sodium carbonate; (5} solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds:; (:7) wetting agents, such as, tor example, cetyl alcohol and glycerol monostsarate; (!) absorbents, such as kaolin and bentonite clay; fb) hihricanis, such a talc, cafcium stearate, magnesium stearate, sohd polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the phamt&amp;ceutleal eompositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers lit soft and hard-filled gelatin capsules using Such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral admuistradoft ioc&amp;de pfeamiaeeuti^Ily acceptable enmlsiops, microemuisions, solutions,. suspensions. syrups, andelixirs, in addition to the active IngTsdieni, fee liquid doxageforms may centals 'kerf dlfoents commonly used in fee-art, .such as water or other solvents, solebilkihg agents ao<| etnulsifiers, such as ethyl alcohol, isopropyl alcohol, efeyl carbonate* ethyl acetate. bengyl alcohol, bermyl bengoate, propylene glycol, 1 ,d~butylene glycol, oils (in particular, cottonseed, groundnot, corn, germ, oli ve, pastor, and sesame oils), glycerol, ietmhydmferyl alcohol, polyefey tone glycols and tatty- acid esters of sofbitan, and mixitires thereof Besides inert diiaenps, fee oral conipositioris can also include adjuvanis such as wetting agents, emulsifying and suspending agents, sweetening, Savoring, coloring, petfeming, and pmservat i w agents.

Suspensions, in addition to fee active compounds, may contain suspending agents such as efeoxylaled isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan: eaters, mierocrystalliue cellulose, aluminum metahydroxide, hentoniies agm^garandfeapeanfe, and mixtures thereof.

Certain compositions disclosed herein may be administered topically, either to skin or to mucosal membranes. The topical formulationsmay further Include one or more of fee Wide variety of agents Inonm fe be effective as sfcm or stiatnm eomeum peoefeehnn enhancers. Examples of these are 2-pyrrolidone, N-mefeyE2-p>Trolidone, diniefeylacefenricfe, dimcfeyifbrmamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and aaone. Additional agents may further be included to nrake fee formulation copre&amp;ally aoeeptahle. Examples of these are fats, waxes, oils, dyes, fragranees, preservatives, stabilisers, and surface active agents. Keratolybe agepfe such as those known In fee art may also he included. Examples are salicylic acid and sutiur.

Dosage ferms for the topical or transdermal administration include powders, sprays, ointments, pastes, Cfearofyloions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions wife a pharmaceutically acceptable carrier, and wife any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition tonsidyest compound of fee invention (e.g,, an ActBII polypeptide), excipients, such as animal and vegefebi© fats, oils, waxes, paraffins, starch, tragacanth, celhdose derivatives, polyethylene glycols, silicones, benfonites, silicic aeM, talc and xine oxide, or mixtures thereof

Powders and sprays can..contain, is addition to a .^l>jeci compi>«nd^ excipients such as lactose» tale, silicic acid* aluminum hydroxide, calcium silicates, and polyamidepowder, or mixtures of these substances. Sprays can additionally contain customary propellants» sechras ctoloroilttorohydrocarbons: and volatile umubstituted hydrocarbons» such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRII polypeptides in combination with one or more pharittaceutically acceptable sterile isotonic aqueous or noiKqneoas solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into: Sterile injectable solutions or dispersions just prior to use, whiuh may OOniain anhoxidautSy buffers, baeteriostais, solutes which render the formulation isotonic with the blood of the tniended recipient or suspending or thickening agents. Examples: of suitable aqueous and nonaqueoas carriers which may be employed in the pharmaceutical: compositions of the ίί^η^οη^Λι^'^ϊδ^',^ίΙ^βοΙ, polyols (such as glycerol, pmpyiene glycms polyeil^lens glycol, and the dike},,and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle si^mti&amp;d&amp;sirpf dispersions, and by the use of surfactants.

The compositionsof the invention may also contain adjuvants, simh as preservatives, wetting agents, emulsifying agents and dispersing agents. Froventidn of timaetion of microorganisms may he ensured by the inclusion of various antibacterial and antifungal agents, tor example, paraben, chlorobutanoi, phenol sorbic acid, and the like, it may also be desirable to include isotonic agents, such as sugars, sodium ohldritie, and the like mto the compositions. M addition, prolonged absorption of the itgeciabie pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. ft is understood that the dosage regimen will be determined by die attending physician considering various factors which modify the action of the subject compounds of the invention (mg,, ActRII polypeptides}. The various factors include. but are not limited to* amount of hone weight desired to he formed, the site of bone damage, the condit ion of the damaged bone, dm sine of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any inaction, time of administration, and other clinical factors. Optionally, the dosage may vary with the type of matrix used m the reconstitution and the types of compounds m the composition. The addition of other known growth factors to the final composition, may also effect the dosage. Progress can be monitored by petiodio assessment of bone growth and/or repair, for example, X-rays, M#orhot|feometric determinations, and letracyd me labeling.

In certain embodiments of the invention, one or more AetRII polypeptides can be: administered, together (simultaneously). or at different times (sequentially or overlapping). 1« addition, AetRII polypeptides can be administered with another type of therapeutic agents, ibr example, a cartilage-inducing agent, a bone-inducing agent, a mnsele-inducingagenl, a fat-reducing, or a nenromimiiieing agent The two types of compounds may be administered simultaneously of at different times,. It is expected that fee AetRH polypeptides of the invention may :fetil n cpnsert wife or perhaps synergistteally with anotlm therapeutic agent.

In a specific example, a variety oC ogteogeniCj carMlage-mdiicing and hone-induemg factors have been described, particularly bisphosphonates. See e.g,, European Patent Application. Nos. 148,155 and 169,0¾ For example, other factors-.feat can be combined with the subject AetRII polypeptides include various growth factors such as epidermal growth factor .(EOF), platelet derived growth factor {RB&amp;Rfi transforming growth factors (TOF-feand TGF-β), and insulin-like growth factor (IGF),

In certain embodiments, fee present invention also provides gene therapy for the in vip: production of AetRII polypeptides; .Such therapy would achieve its therapeutic effect, by : introduction of fee ActRH polynucleotide sequences into ceils or tissues having the disorders as listed above. Deli very of AetRII polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Preferred for therapeutic delivery of AetRII polynucleotide sequences is fee use: of targeted liposomes.

Various feral vectors which can he utilised forgene therapy as taught herein include adenovirus, herpes vims, vaccinia, or, preferably,an RNA vims such as a retrovirus. Preferably, the retrovfefi vector is a derivative of a murine or avian retrovirus. Examples of mmovirai vectors in which» single foreign gene can fee Inserted include, but are not limited to; Moloney murine leukemia vims iMoMuI.V}, Harvey murine sarcoma virus (HaMuSV), murine mammary tumor vims (MuMTV), end Rous Sarcoma Virus: (RS¥). A number of additional retroviralveeterspatt incorporate mollipie genes, Alt of these vectors can transfer or incorporate a gene for a sekctsbk market so that transduced ceils can he identified and geseraied. Eeteovkal vectors can be made ter get-specific by attaching, for example, a lugar* a glycolipid, or a protein. Preferred taxgetmg is accompitshod by using an antibody. Those of skill· in the art will fecogpissd that specific polynucleotide 'Sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ActKii polynucleotide. In one preferred embodiment, the vector'is targeted to bone, cartilage, muscle or neuron eells/tissues.,

Altematwely, tissue culture cells can be directly transfected wife plasmids encoding the mtevdraf siructnial geaes gag, pel and env, by conventional calcium phosphate transfcetkm» These cellsare then transfected with the vector plasmid conteming the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Ajintfe fergeled delivery system tor ActEH nolynucfeotides is a eolloidal dispersion system. Colleirfel dispersion systems include macromoleeule complexes, nanocapsules, nimresphetes, heads, and hpfofogsed systems including oil-in-water emulsions, micelles, mixed micelles, and .liposomes. The preferred colloidal system of this invention is a liposome. liposomes are adifieiai membrane vesicles which are useful as delivery vehielcs in vitro and in vivd,·-KiHAs,.δ$£&amp;·snd'mta&amp;fviriofisi can be encapsulated within the aqueous interior and be delivered te cells in a biologically active form (see e,;g.s Fraley, et at, Trends Eiochem, Sci., 6:77, 39bl}:. Methods : for efficient gene transfer: using a liposome vehicle, are known in the art, see e.g., Mannino, et at,, Bieteehniques,. 6:682,1988. The composition of the: liposome is usually a combination, of phosphUlipids, usually in eombraabon with steroids, especially chotesterol Otlisr phospholipids Of bitter lipids may also be used. The physical eharactemiics of liposomes depend on pH, ionic strength, and fee presence of divalent cations.;

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglyeefol, phos|feaddytehoiiue, phosphaiidylserine* phosphatidyleihanolatnirie, sphiogolipids, eerehrossdes, and gangliosides. Illustrative phospholipids include egg phosphatidylehclme, dipalafeoyfphesphatidylcholme, and dlstrmmylphosphatidyteholme. The targeting ofliposomes is also possible based on, for example. Organ-specificity, cell-specificit>f, and organelle^eciiscity and is known in the art.

EXEMFLiFICATIDH

Use invention now being generally described, it will be more readily understood by reference to the fallowing examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention, and are not. intended to limit the invention.

Applicants generated a series of mutahoim inihe extracellular domain of ActRiffi and prndneed these mutant proteins as soluble fusion proteins between exfeneellulat AetRIIB and an Fc doshalii A co-crystal structure of Activin and extracellular AelRIIB did not show any role for the final (C-terminal) 15 annuo: acids (referred to as the “fair herein) of the exlraeeifular domain in ligand binding. This sequence failed to resolve on the crystal structure, suggesting that these residues are present in a flexible loop that did not pack uniformly fe the crystal. Thompson' et ai EMBG J. 2001 Apr I;22(7)'1555-66. This sequence: is also poorly conserved between AetRJiB and AetRlfA. Aeeordingly, daese residues were omitted in foe basic, or background, ActRIIB-Fc fusion construct Additionally, position 64 in the background form is occupied by an alanine, which is generally considered the ‘‘wild type*' Tours, although a A64R allele occurs naturally. Thus, the background AefEIiB-Fo fusion has the sequence (Fc portion underimedKSEQ ID NO: 14): SGRGBAEXRBOYYNANWEiJjRT^

KOOyDKDFNO¥DRDECVATEENFO¥¥FC€CEtlhlICNERFTMLPEAGdQlTTr€FPCF

Sufprisingiyj as discussed below, the Gdernsnal tail was fbuird to enhance acbvih and 0PM1 hinding, thus a preferred version of AetROB-Fe has a sequence (Fo portion : under! i ned)(SEQ ID 'NO:! 5): SGRGEAETREGl¥¥NANWELEKTNQSGLER0E6EQDKRLB0¥ASWANSSG'TiELVK:

KGCWLDDFNGYDRQECVATEENEQVYfCCGEGNFCNERrrHLPEAOGFEVTYEFFE TAPTGGeTm>CPF0FAFELLGGFS¥Ff.EFFKPKDTl,Ml:SRTPE¥B2W¥D¥SBEDPi¥

KMWY¥DGVBVHmKTEFRBIO¥MSyyRV¥SVLTVLHODWmGKEm:;K:VSH$;A

GPBmyKTYFFyLDSDGSFFL¥^KOymSRWOOGNVFS€syMH£ALHNHyiX>KSL

StMGK

Various mutations wete iniriviucsdinio she 'background ActftUB-Fc pmtdfev Mmations were generated in ActRHB extracellular domain by PCS mutagenesis. After ,PCR* fragments were purified 'thru Qiagen column,: digested wish Sfoi and Age! and gel phrifted. These ftspsents were ligated into eripmsribn vector pAff>4 such that upon Hgaiion it created fusion chimera with human IgG 1, Upon transformation into E. noli DHS alpha, colonies were picked and DMAs were isolated. Alt nnitahts Were sequence wiled.

Ail of the : mutants were produced in ΗΕΟΡ31Γ ceils by transient transfehon. In summary, in a SQOrol spinner.. ΒΕΪ093Τ cells were set up at :ftxt0s eells/ml in Freestyle (Invinogen} media in 250ml volume and grown overnight;, Next day* these cells were treated with DMA: PEI (!;!) complex at 0,5 ug/mi tinal UNA concentration. After 4 hrs, 250 ml media was added and eels were grown tar 2 days. Conditioned media was harvested by spinning down the eels and concentrated.

All dre mutants' were purified over protein-A column and eluted with low pH (3.0) glycine buffer. After neutralization, these were dialyzed against BBS.

Mutants were also produced in CHO cels fey similar methodology. / ...

Mutants were tested in binding assays and bioassays described below; Proteins expressed in CHO cels and HEK293 ceils were indistinguishable in the binding assays and bioassays.

Example 2. (IDF-11 and Activin A Binding

BindingAetMIBT%proteins was tested in a Bia€ore?M assay. GDF-tl or Activin A (“ActA”) were immohltned odd BiaCore CMS chip using standard amine coupling procedure, The ActRlIB-Fe mutant Or wild-type protein was loaded onto tire system;, and binding was measured. Results are surmnariged in table i* below.:

Table T SoluMe AetRIlBdm binding to GDFi 1 and; Activin A (BlaCore assay)

* No observed binding - < 1/5 WT binding

- - 1/2 WT binding + WT 4+ < 2x increased binding •Ή-*- ~5χ increased binding 4..5.44. „4 qx increased binding 444^4,., 40·^ incrsased binding

As shown in Table 1, mutations had vs^ing ei&amp;cM mligand binding, Ίhe addition of the Cderminal I S amino aetis of the exiraceliular domain, ««used a substantial increase in binding affinity for both ActiWn A and OpF~l 1, and ilk expected that Sik effect will translate to essentially all of the other mutations. Other mutations caused anoverall increase in ligand binding affinity, Including the naturaliy occurring allele A64E and K74Ai The R40A mui8%S eatssed a moderate decrease in binding aftinny for both Activin A and CDF-! I, 'Many mntaHons aboltslwd detecfohlb binding to AeftvinA and G0F~1Ϊ, including; Ε37Λ. E56ArW7SA, DESK, D80R, D80A, DSOG, D80F, DSOM and D80N. Certain mutations caused a shift in selectivity, The following mataftoas1 caused an increase in fhs ratio of 0DF41 toAetNn A binding:1£?4YS X74F, K174I .and D80L The following mutations caosed a decrease In foe ptio of GDP-11 to ..Activin A binding: D54A, &amp;55A, L79Aand F82A. ^Exa^^.a,...BbassaytfeGDP->il aitd Actferoae^ted signaling.

An A-204 Reporter Gene Assay was used to evaluate the effects Of ActRriB-Fe proteins on signaling by GDF- II and Aoftvin A· Cell Hoe: Human Rftabdomyosareoma (derived from muscle). Reporter vector: pGOfGAG A)12 (Described in Dennler et a I, i 99% EMBO 17: 3091-31Q§.) See Figure 5. The GAGA12 motif is present in TGF-Beta responsive genes ( PAH gene) , so this vector is of genera! use for factors signaling through SmsdS and 3,

Day I: Split A-204 cells into 48-svcil plate.

Day 2: A-204 cells transfected with 10 eg pGL3(CAGA)i2 nr pGL3(CAGA)l2(10 ag)a· pBLCM Y |1 eg) and Fngene,

Day 3: Add factors (dilated1 info mediunG· 0,1 % EtSA). Inhibitors need to fee pre incubated with Factors for 1 hr before adding to cells, 6 hrs later, ceils rinsed with PBS, and lyse cells,

This Is followed by a Lecfterase assay. In the abscuce of any inhibitors, Activin A showed 10 fold stimulation of reporter gene expression and ah BDdff ^ 2 ng/ml. GHF-l1: Id fold stimulation, B05O: - L.S ng/asL

As shown in Figure 16, wild-type (bacligronnd A64):A«^®f^C'im¥bitS'G-DF~ll signaling in foe A-204 Reporter Gene Assay. The background A64 construct showed an iolhbifory eiieet on GDF~11 activity, The A64K. mutation (also a naturally occurring form): caused a substendaiinereaseln GDFG Imbibition, and a combination of the A64R mutation with the addition of foe 15 G-terminal amino acids of foe extraoellnlar domain (foe I S amino acid ‘‘fair), produced an even mere potent inhibitor of GDF~ l l acti vity. As shown in Figure !7, the %ekgfound AE4 cmisimet showed aninhibitory effect on Activio A activity. The·. K^A ffiatatioR caused a MbsM«ti^I mcrease1» Actwki A inhibition, A control sample lacking Aetivin A showed no activity.

These data from the well. binding assays shown in

Table 1 and demonshaie thaf the effects of the swioas mntations translate to a. biological system.

Example 4: AetRIlA^Fc Fusion Pn>fosns

As shown in Figure 34, AeiRjlA and AetRlIB ate highly conserved. Aecdr&amp;iglyimost of the rnhtations testedih AetMXB are expeeted to have similar effects in AefRllA. Thus, a background ActKHA-Pc fusion may he constructed with the following sequence (Fc portion imder!ined)(SEQ ID NO: 16):

AfEC3RSETQE€iLFFiAAlTWHKfiliThl^TQ^lF€Y<3DiiBKRKHl3EAT^KhJiSi3SiEIVKQ eCWEDBmCYDRTDCVEiroSFE WFCGGEShfMCiVE^SYFPEMeMTroZEZA FELLSGFSWi^PFK^ICBTfcMfSRTFEYTGVYAnDYSHEDFEyKFMWYVDGVEVfiHAK TOREEGY^STYRVYSYLTYLHGDWhNGKEYRCKySNKAEFVFIEKTlSKAKGGFRE

As discussed below, the C-teiminal tail was found to enhance achy in and GGF-li binding, thus a preferred version of AciMiA-Fe has a sequence (Fc portion underhnedf(SEQ I0HO:!?):

AITGRSiTQBGlFFNANWEKDRT^QXGYEPCYGDICGK^HGEATWKixISGSIEliVKQ

0CWEP0HGYORTDCYEKICDSFEYYFCC€EGNMChlEICF8YFPEMEYTQI^SMFVTF

KFEGGGTHT€FFCFAPELLGGPSWLFP»KDTLMISRTPi¥TCV¥¥DVSHEPF£¥K

FWY¥DG¥E¥^AKIKPFEEOYNSi¥R¥VSVLT¥l,HGDWmGKEYICGK:¥ShrRAE

P¥ldEK'ifSMKGOPREPO¥YlLPPShEEMTKNO¥SETClYRGF¥ESBlA¥EWESNGD

i:fodNT^YRT'rPF¥LDSD(foFFi...YSKLT¥DKSRWOOGN¥FSCS¥,'MH.EALHNHYTOKSI.S

ESPGK to those made in ActRIIB, may be made hi the hhekgK>wnd vaAioB of AetMiA tji tJ\e “iair’ yeisioo of ActRIiA. lie oomsspondgsce betwe^n ActRIlB and Aet&amp;llA imitations is shown in Table 2 below.

mcmxmmKM by reference

Al! publications andpatents mentioned herein are hereby incorporated by reference in their entirety as Reach individual publicationorpatent w# speeifieai!y and indm^pally indicated to be ineorprated by reference.

Wbtt&amp;lpedBe .embodlmfenfs. of the subject matter have been discussed, the above specification Is illustrative and not restrictive. Marty variations will become apparent to those shilled in the art upon review of ibis specification and the claims below,: The lull scope of the : invention should be determined by refc^pe to the claims» along with their fell scope of equivalents» and the specification, along with sueis variations.

Claims (19)

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

   1. An ActRIIB polypeptide comprising an extracellular domain of an ActRIIB polypeptide having an amino acid sequence that is at least 85% identical to SEQ ID NO: 2, a flexible linker having the sequence TGGG, and an immunoglobulin Fc domain, wherein the immunoglobulin Fc domain is fused to the C-terminus of the extracellular domain of the ActRIIB polypeptide by the flexible linker, and wherein the extracellular domain of the ActRIIB polypeptide comprises one or more mutations at a residue selected from: L79, D80, and F82 using the amino acid numbering of SEQ ID NO: 4.
2. 2. The ActRIIB polypeptide of claim 1, wherein the amino acid sequence of the extracellular domain of the ActRIIB polypeptide is at least 90% identical to SEQ ID NO: 2.
3. 3. The ActRIIB polypeptide of claim 1, wherein the amino acid sequence of the extracellular domain of the ActRIIB polypeptide is at least 95% identical to SEQ ID NO: 2.
4. 4. The ActRIIB polypeptide of claim 1, wherein the amino acid sequence of the extracellular domain of the ActRIIB polypeptide is at least 99% identical to SEQ ID NO: 2.
5. 5. The ActRIIB polypeptide of any one of claims 1-4, wherein the immunoglobulin Fc domain comprises the amino acid sequence of SEQ ID NO: 13.
6. 6. The ActRIIB polypeptide of any one of claims 1-5, wherein the polypeptide comprises a L79 mutation, using the numbering of SEQ ID NO: 4.
7. 7. The ActRIIB polypeptide of any one of claims 1-5, wherein the polypeptide comprises a D80 mutation, using the numbering of SEQ ID NO: 4.
8. 8. The ActRIIB polypeptide of any one of claims 1-5, wherein the polypeptide comprises a F82 mutation, using the numbering of SEQ ID NO: 4.
9. 9. The ActRIIB polypeptide of any one of claims 1-8, wherein the one or more mutations in the extracellular domain of the ActRIIB polypeptide alter binding of one or more ActRIIB ligands to the ActRIIB polypeptide.
10. 10. The ActRIIB polypeptide of claim 9, wherein the one or more ActRIIB ligands is selected from: activin, BMP7, GDF8, GDF11, and Nodal.
11. 11. The ActRIIB polypeptide of any one of claims 1-10, wherein the polypeptide comprises one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
12. 12. A pharmaceutical preparation comprising: i) the ActRIIB polypeptide of any one of claims 1-11; and ii) a pharmaceutically acceptable carrier.
13. 13. The pharmaceutical preparation of claim 12, wherein the preparation is substantially pyrogen-free.
14. 14. An isolated polynucleotide comprising a coding sequence for the ActRIIB polypeptide of any one of claims 1-11.
15. 15. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 14.
16. 16. A cell comprising the recombinant polynucleotide of claim 15.
17. 17. The cell of claim 16, wherein the cell is a mammalian cell.
18. 18. The cell of claim 17, wherein the cell is a human cell.
19. 19. A method of making an ActRIIB polypeptide, comprising: a) culturing a cell under conditions suitable for expression of the ActRIIB polypeptide, wherein the cell comprises the recombinant polynucleotide of claim 15; and b) recovering the ActRIIB polypeptide so expressed.