USOO8796199B2
(12) United. States Patent (10) Patent N0.: US 8,796,199 B2 Grinberg et al. (45) Date of Patent: Aug. 5, 2014
(54) USES OF CERBERUS AND DERIVATIVES WO WO-02/078516 10/2002 THEREOF W0 wo-02/090992 11/2002 WO WO-03/012082 2/2003 . _ WO WO-03/055443 7/2003 (75) Inventors: Asya Grinberg, Boston, MA (US), John W0 WOW/055911 7/2003 Knopf, Carllsle, MA (Us); Ravmdra W0 wo-03/072714 9/2003 Kumar, Acton, MA (US); Jasbir W0 W0 03/106657 12/2003 seehra, Lexington, MA (US) WO WO-2004/074460 9/2004 WO WO-2005/003158 1/2005 (73) Assignee: Acceleron Pharma, Inc., Cambridge, W0 WO'ZOOS/115439 12/2005 MA US ( ) OTHER PUBLICATIONS ( ) NOtlce' sub?qw any (31531211111651 the fiermef?glg The Chimpanzee Sequencing and Analysis Consortium. Sep. 2005; gage? 11552226113 6 880;; JuSte un er Initial sequence of the chimpanzee genome and comparison with the ' ' ' ( ) y ays' human genome. Nature. 437:69-87.* _ Avsian-Kretchmer et al., “Comparative geneomic analysisof the (21) Appl' NO" 12/940’740 eight-membered ring cystine knot-containing bone morphogenetic _ _ protein antagonists,” Molecular Endocrinology, 18(1): 1-12 (2004). (22) Flled' NOV“ 5’ 2010 Belo et al., “Cerberus-like is a secreted factor with nerualizing activ _ _ _ ity expressed in the anterior primitive endoderm 0f the mouse gas (65) Pnor Pummatlon Data trula,” Mechanisims of Development, 68:45-57 (1997). Us 2011/0046057 A1 Feb' 24’ 2011 Biben et al., “Murine Cerberus Homologue mCer-l: A canadidate anterior patterning molecule,” Developmental Biology, 194: 135 -151
_ _ (1998). Related U-s- APPhcatlon Data Bouwmeester et al., “Cerberus is a head-inducing secreted factor (62) Division of application No. 12/001 ,494, ?led on Dec. ggfzsgsggagngnor endOderm Ofspemann S orgamzer’ Namre’ 10’ 2007’ HOW pat NO' 7’833’97 1' BrecherA.S., et al., “Acetaldehyde Inhibits Chymotrypsin and Serum (60) Provisional application No. 60/873,933, ?led on Dec. Anti-ChYIHOtYYPSiIl ACtiVity,” J~ Investig Med» 46(4)1146-152 8’ 2006' (1998). Abstract only. Esteban et al., “The novel Cer-like protein Caronte mediates the (51) Int_ CL extablishment of embryonic left-right asymmetry,” Nature, 401 :243 C07K 14/00 (2006.01) 251 (1999) _ _ _ _ (52) U 5 Cl Katoh et al., “Identi?cation and characterization of human U'Séc ' 514/1_ 530/350 CKTSFlBZ and CKTSFlBS genes in silico,” Oncology Reports, I ...... , 121423427 (2004), (58) Fleld 0f ClaSSI?catlon seaTCh Kuroda et al., “Neural Induction in Xenopus: requirement for USPC ...... ' ...... 514/1; 530/350 extodermal and endomesodermal Signals Via Chordin, noggin, See application ?le for complete search history. ?-catenin and cerberus,” PLoS Biology, 2(5):0623-0634 (2004). Lah et al., “Human cerberus related gene CER] maps to chromosome (56) References Cited 9,” Genomics, 55:364-366 (1999). Livingston, S.F., et al., “The Signi?cance ofChymotrypsin-Inhibitor U~S~ PATENT DOCUMENTS Levels in the Serum of Patients with Carcinoma of the Breast,” 5 935 852 A 8/1999 Follettie et al Cancer Research, 17(9):857-861 (1957). 6’133’232 A 10/2000 De Robenis ét 31 Marques et al., “The activity of the nodal antagonist Cerl-Z in the 636103510 Bl 8/2003 Valenzuela et 31‘ ' mouse node is required for correct L/R body axis,” Genes & Devel 7,316,998 B2 * 1/2008 Knopf et al...... 424/1411 013mm, 181342-2347 (2004) 2002/0164682 A1 11/2002 Follettie et al, Motoko Yanagita. BMP antagonists: Their roles in development and 2003/ 0134790 A1 7/2003 Langenfeld involvement in pathophysiology. Cytokine and Growth Factor 2003/0194704 A1 10/2003 Penn et al. Reviews, 16(3): 309-319 (2005). 8(1) 1(1); Yalenzuteli et 31' Pearce et al., “A mouse cerberus/dan-related gene family,” Develop 2004/0181033 A1 9/2004 $252121? ' mental Biology’ 209198410 (1999) 2005/ 0186663 A1 8/2005 Davies et al. (Continued) 2006/0105376 A1 5/2006 Isogai et al. 2008/0032304 A1 2/2008 Isogai et al.
FOREIGN PATENT DOCUMENTS Primary Examiner * Karen Cochrane Carlson (74) Attorney, Agent, or Firm * Ropes & Gray LLP EP 1347046 9/2003 WO WO-97/48275 12/1997 WO WO-98/34951 8/1998 (57) ABSTRACT WO WO-98/49296 11/1998 W0 WG-99/01553 1/1999 The disclosure relates to Cerberus/ Coco polypeptides or vari ag ants thereof for use in treating a variety of disorders associ W0 WO 02/10214 2/2002 ated With myostatin, nodal and GDP-11. WO WO-02/32929 4/2002 WO WO-02/054940 7/2002 WO WO-02/077204 10/2002 18 Claims, 8 Drawing Sheets US 8,796,199 B2 Page 2
(56) References Cited Silva et al., “Endogenous Cerberus activity is required for anterior head speci?cation in Xenopus,” Development, 130(20):4943-4953 (2003). OTHER PUBLICATIONS Stanley et al., “Murine cerberus homologue Cer] maps to chromo Piccolo et al., “The head inducer Cerberus is a multifunctional some 4,” Genomics, 49:337-338 (1998). antagonist of Nodal, BMP and Wnt signals,” Nature, 397:707-710 (1999). * cited by examiner US. Patent Aug. 5, 2014 Sheet 1 0f8 US 8,796,199 B2
NODAL Figure1 BMP US. Patent Aug. 5, 2014 Sheet 2 0f8 US 8,796,199 B2
RU HSNOcISElH US. Patent Aug. 5, 2014 Sheet 3 0f 8 US 8,796,199 B2
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In CROSS-REFERENCE TO RELATED the myostatin null mice, the animals were signi?cantly larger APPLICATIONS than wild-type mice and had a large and widespread increase in skeletal muscle mass. Furthermore, two breeds of cattle, This application is a Divisional of US. application Ser. No. characterized by increased muscle mass, have mutations in 12/001,494, ?led on Dec. 10, 2007, which claims the bene?t the myostatin coding sequence (McPherron et al., Proc. Natl. of US. Provisional Application No. 60/873,933, ?led on Dec. Acad. Sci. 94:12457-61 (1997)). A naturally occurring myo 8, 2006. The contents of these applications are hereby incor statin reduced-function mutation in a human child is associ porated by reference in their entireties. ated with gross muscle hypertrophy and a family history of exceptional strength (Schuelke et al. 2004 Jun. 24; 350(26): SEQUENCE LISTING 2682-8). An antibody against myostatin is reported to have bene?cial effects in animal models of muscle disorders, The instant application contains a Sequence Listing, which including amyotrophic lateral sclerosis (Holzbauer et al. has been submitted via EFS-Web and is hereby incorporated Neurobiol Dis. 2006 September; 23(3):697-707). by reference in its entirety. Said ASCII copy, created on Nov. Additionally, it should be noted that the serum and intra 4, 2010 is named PHPH024 1 02.txt and is 37,983 bytes in size. muscular concentrations of immunoreactive myostatin are increased in HIV-infected men with muscle wasting com BACKGROUND OF THE INVENTION 20 pared with healthy men, and correlate inversely with the fat-free mass index. These data support the hypothesis that Transforming growth factor-[3 superfamily proteins repre myostatin is a negative regulator of skeletal muscle growth in sent a large family of cytokines that includes the TGF-Bs, adult men and contributes to muscle wasting in HIV-infected activins, inhibins, bone morphogenetic proteins (BMPs) and men (Nestor et al., supra). Mullerian-inhibiting substance (MIS) (for review, see Mas 25 In view of the above ?ndings, a need exists for a manner of sague et al., Trends Cell Biol. 7:187-192, 1997). These pro regulating myostatin activity, particularly in individuals who teins contain a conserved C-terminal cysteine-knot motif, and experience muscle wasting as a result of a condition or dis serve as ligands for diverse families of plasma membrane ease state such as, for example, aging related frailty, cachexia receptors. Members of the TGF-[3 family exert a wide range in Autoimmune De?ciency Syndrome (AIDS), Multiple of biological effects on a large variety of cell types. Many 30 Sclerosis, muscular dystrophy, ALS and cancer-cachexia. members of this family have important functions during The present invention provides methods and compositions embryonal development in pattern formation and tissue which may be utilized to help individuals with such muscle speci?cation; in the adult, these factors are involved in pro wasting conditions and provides further insight into the regu cesses such as tissue repair and modulation of the immune lation of myostatin gene expression. system. 35 Activities of the TGF-[3 superfamily proteins are regulated SUMMARY OF THE INVENTION through various means. One of the negative regulations for the BMP subfamily of proteins is through a relatively large In part, the disclosure relates to the discovery that two family of so-called Bone Morphogenetic Protein (BMP) human proteins, Cerberus and Coco, that belong to a group of antagonists/repressors. These BMP repressors represent a 40 GDF/BMP antagonists, bind to and antagonize myostatin, subgroup of proteins that bind BMPs, and interfere with BMP GDF11 and Nodal, and furthermore, that the myostatin/ binding to their membrane receptors, thereby antagonizing GDF11 binding domain resides in the cysteine knot domain their actions during development and morphogenesis. of these proteins. Furthermore, with respect to Cerberus, The BMP repressors can be further divided into three myostatin/GDF11 binding and antagonist activity can be groups of proteins based on structural analysis, especially the 45 separated from the BMP4/2 binding and antagonist activity. number of structurally conserved Cys residues in their C-ter Therefore, the disclosure provides, in part, methods for minal characteristic “Cys-knot” structures: the 8-, 9-, or antagonizing myostatin and GDFl 1 in vivo by administering 10-member ring Cys-knot BMP repressors. The 8-member polypeptides comprising a myostatin binding portion of Cer ring (CAN subfamily) repressors can be divided further into berus or Coco, or variants thereof. One aspect of the invention four subgroups based on a conserved arrangement of addi 50 provides polypeptides, and pharmaceutical preparations tional cysteine residuesigremlin and PRDC, Cerberus and thereof, of Cerberus, Coco (from human or non-human ani coco, and DAN, together with USAG-l and sclerostin. mals) or a derivative thereof (collectively herein “Cerberus/ Orthologs of these human BMP antagonists in the genomes of Coco proteins”) for inhibiting the function/ signaling of several model organisms have also been identi?ed, and their Nodal, myostatin, GDF11 and, in certain forms, BMP4 and/ phylogenetic relationship has been analyzed (Avsian-Kretch 55 or BMP2. In certain embodiments, preparations of the subject mer and Hsueh, Mol Endocrinol. 18(1): 1-12, 2004, incorpo Cerberus/Coco polypeptides may include variant Cerberus or rated herein by reference). Coco proteins that retain all or a substantial portion of the Myostatin, or growth/differentiation factor 8 (GDP-8), binding af?nity of the parent protein to Nodal, myostatin, also belongs to the transforming growth factor-[3 (TGF-B) GDF11 and/or another BMP (such as BMP4). In certain superfamily (McPherron et al., Nature 387:83-90 (1997)). 60 embodiments, preparations of the subject Cerberus/Coco The human myostatin gene has been cloned (Nestor et al. polypeptides include variant Cerberus or Coco proteins that Proc. Natl. Acad. Sci. 95:14938-43 (1998)), and it has been retain all or a substantial portion of the binding af?nity of the reported that myostatin immunoreactivity is detectable in parent protein to myo statin and/ or GDF11 while eliminating human skeletal muscle in both type 1 and type 2 ?bers. With or reducing binding to BMP4 and/or BMP2. In certain respect to function, myostatin may play a role in negatively 65 embodiments, the disclosure provides the observation that regulating the growth and development of skeletal muscle full-length human Cerberus is unstable in the presence of (Nestor et al., supra). human serum, and thus altered forms of Cerberus (both US 8,796,199 B2 3 4 BMP4 binding forms and selective myostatin/GDFl 1/Nodal syndrome, muscular dystrophies, neuromuscular diseases, binding forms) may be prepared that are stable in the serum motor neuron diseases, diseases of the neuromuscular junc for a period of at least 24 hours, and optionally 2, 3, 5, 7, 14 tion, and in?ammatory myopathies. or 21 days or longer. This observation may be extrapolated to In certain embodiments, the mysotatin inhibitor is a Coco, and thus altered forms of Coco may be prepared that are polypeptide that includes a myostatin binding domain of a stable in the serum for a period of at least 24 hours, and Cerberus/Coco protein. For instance, the Cerberus protein optionally 2, 3, 5, 7, 14 or 21 days or longer. In certain variant can be derived from a human, mouse, or other species embodiments, the disclosure provides pharmaceutical prepa of Cerberus, including a human or mouse Cerberus variant rations for inhibiting myostatin, comprising a myostatin sequence sharing at least about 50%, 60%, 70%, 80%, 90%, antagonist protein that includes (at least) a myostatin binding 95%, or 99% or more sequence similarity or identity with the domain of a Cerberus/Coco polypeptide or variant thereof. human or mouse Cerberus protein, and substantially retain The myostatin antagonist protein binds to and neutralizes one the binding af?nity of wild-type Cerberus for myostatin. or more of nodal and/or myostatin. Preferably, the pharma Likewise, the Coco protein variant can be derived from a ceutical preparation is substantially free of pyrogenic mate human, mouse, or other species of Coco, including a human rials so as to be suitable for administration as a human or or mouse Coco variant sequence sharing at least about 50%, veterinarian therapeutic. 60%, 70%, 80%, 90%, 95%, or 99% or more sequence simi Myostatin is widely recognized as an antagonist of muscle larity or identity with the human or mouse Coco protein, and growth. Furthermore, myo statin null mice have shown resis substantially retain the binding af?nity of wild-type Coco for tance to obesity and diabetes under certain conditions. There myostatin. fore, the Cerberus/Coco proteins and pharmaceutical prepa 20 In certain related embodiments, the mysotatin inhibitor is a rations described herein can be used to reduce the severity of polypeptide that includes a myostatin binding domain of a a pathologic condition, which is characterized, at least in part, Cerberus/Coco protein, which polypeptide does not substan by an abnormal amount, development or metabolic activity of tially bind BMP-4 or BMP-2. For instance, the myostatin muscle or adipose tissue in a subject. For instance, the phar binding domain can be derived from a human, mouse, or other maceutical preparations of the present invention can be 25 species of N-terminally truncated Cerberus, including a administered in an amount effective to prevent, ameliorate or human or mouse Cerberus derivative, with amino acid resi reduce the severity of a wasting disorder, such as age-related dues starting from any one of residues 106-1 19 of SEQ ID No. wasting, age-related frailty, cachexia, anorexia, Duchenne 1 or 2, and ending at any residue after residue 241 of SEQ ID Muscular Dystrophy (DMD) syndrome, Becker’s Muscular No. 1 or 2, preferably ending at any residue between residues Dystrophy (BMD) syndrome, facio-scapular-humeral (FSH) 30 241 and 267 of SEQ ID No. 1 or 2 (all residue numbers muscular dystrophy, other muscular dystrophies, AIDS wast inclusive). ing syndrome, neuromuscular diseases, motor neuron dis For example, residues 106-119 of human Cerberus are: eases, diseases of the neuromuscular junction, and in?amma tory myopathies. Excessive BMP4 activity has been associated with pathological ossi?cation of various connec 35 PPGTQSLIQPIDGM (SEQ ID NO: '7) tive tissues. Therefore, the Cerberus/ Coco proteins and phar maceutical preparations that retain anti-BMP4 activity can be Residues 241-267 of human Cerberus are: used to reduce the severity of a pathologic condition, which is characterized, at least in part, by an abnormal ossi?cation in CKVKTEHEDGHILHAGSQDSFIPGVSA (SEQ ID NO: 8) tissues such as muscles, tendons, and ligaments. BMP4 is also 40 associated with Osteoarthritis (OA), including the develop Also included are Cerberus derived variant sequences, e. g., ment of osteophytes and synovial thickening; Fibrodysplasia an N-terminally truncated myo statin binding domain of Cer ossi?cans progressiva (FOP); and atherosclerosis, especially berus that retains myostatin binding activity but loses other in?ammatory response in early steps of atherogenesis in BMP binding activity. Variant sequences may be desirable as lesion-prone areas; and craniosynostoses. Nodal signaling 45 a way to alter selectivity of the inhibitor (e.g., relative to has been associated with certain cancers, particularly mela GDP-8, GDP-1 1 or nodal binding), alter other binding char noma. Accordingly, Cerberus/Coco proteins and pharmaceu acteristics with respect to myostatin (such as Kd, and/or K0” tical preparations that retain anti -Nodal activity can be used to or Kofrates), or improve biodistribution or half life in vivo or treat tumors, particularly tumors such as melanomas in which on the shelf. Nodal participates in tumor growth and development. 50 In certain preferred embodiments, the Cerberus polypep Another aspect of the invention provides a pharmaceutical tide (full-length or N-terminally truncated) comprising the preparation of Cerberus/Coco protein derivative for speci? myostatin binding domain binds myostatin with a Kd of 1 uM cally inhibiting Nodal and/ or myo statin function without sub or less, and more preferably a Kd of 100 nM, 10 nM or even 1 stantially compromising BMP (such as BMP-4) signaling nM or less. (e. g., does not substantially bind BMP-4 or other BMPs). 55 In certain related embodiments, the mysotatin inhibitor is a Exemplary preparations of this aspect of the invention polypeptide that includes a myostatin binding domain of a include polypeptides including the N-terminal truncated ver Coco protein, such as the human Coco protein shown in SEQ sions of Cerberus or Coco, or other fragments that include the ID N015 or in GenBank Accession number 22749329. cysteine-core. These so-called “N-terminally truncated Cer In certain preferred embodiments, the Coco polypeptide berus/ Coco derivatives” can be used to reduce the severity of 60 (full-length or N-terminally truncated) comprising the myo a pathologic condition, which is characterized, at least in part, statin binding domain binds myostatin with a K d of 1 uM or by an abnormal amount, development or metabolic activity of less, and more preferably a Kdof 100 nM, 10 nM or even 1 nM muscle or adipose tissue in a subject. For instance, the phar or less. maceutical preparations of the present invention can be In certain embodiments, the Cerberus/Coco polypeptide administered in an amount effective to prevent, ameliorate or 65 (e.g., a myostatin binding domain thereof) is part of a fusion reduce the severity of a wasting disorder, such as cachexia, protein including, in addition to the myostatin binding anorexia, DMD syndrome, BMD syndrome, AIDS wasting domain, one or more polypeptide portions that enhance one or US 8,796,199 B2 5 6 more of in vivo stability, in vivo half life, uptake/administra variant Cerberus/Coco polypeptide, as described herein, and tion, tissue localization or distribution, formation of protein a label or instructions for use in promoting growth of muscle complexes, and/or puri?cation. For instance, the fusion pro tissue in a human patient. tein can include an immunoglobulin Fc domain. The fusion Still another aspect of the disclosure relates to packaged protein may include a puri?cation subsequence, such as an pharmaceuticals comprising a pharmaceutical preparation of epitope tag, a FLAG tag, a polyhistidine sequence, or as a a variant Cerberus/Coco polypeptide, as described herein, GST fusion. and a label or instructions for veternerian use in promoting In certain embodiments, the Cerberus/Coco polypeptide growth of muscle tissue in a non-human mammal. (e.g., myostatin binding domain thereof) is part of a protein Another aspect of the disclosure relates to a method for inhibiting myostatin signal transduction in vivo by adminis that includes one or more modi?ed amino acid residues, such tering a pharmaceutical preparation of one or more of the as a glycosylated amino acid, a PEGylated amino acid, a subject variant Cerberus/Coco polypeptides. The subject famesylated amino acid, an acetylated amino acid, a biotiny method can be used to promote muscle growth, promote lated amino acid, an amino acid conjugated to a lipid moiety, adipogenic differentiation, and/or promote bone growth or or an amino acid conjugated to an organic derivatizing agent. mineralization in human patients or in non-human animals. In certain embodiments, a subject variant Cerberus/Coco In certain embodiments, the treatment methods of the polypeptide is selective for binding and inhibition ofmyosta present disclosure can be used to reduce the severity of a tin, e.g., relative to GDF-ll and/or nodal. For instance, the pathologic condition, which is characterized, at least in part, variant Cerberus/Coco polypeptide can be one which has a by an abnormal amount, development or metabolic activity of dissociation constant (K) for myostatin binding that is at 20 muscle or adipose tissue in a subject. For instance, the phar least 2 times less than its Kd forbinding GDF-ll and/or nodal, maceutical preparations of the present disclosure can be and even more preferably at least 5, 10, 100 or even 1000 administered in an amount effective to prevent, ameliorate or times less. Whether by virture of binding kinetics or biodis reduce the severity of a wasting disorder, such as cachexia, tribution, the subject variant Cerberus/Coco polypeptide can anorexia, DMD syndrome, BMD syndrome, AIDS wasting also be selected based on relative in vivo potency, such as an 25 syndrome, muscular dystrophies, neuromuscular diseases, inhibitor that has an EC50 for inhibiting myostatin activity, or motor neuron diseases, diseases of the neuromuscular junc a particular physiological consequence (such as promoting tion, and in?ammatory myopathies. muscle growth) that is at least 2 times less than its EC50 for Exemplary muscular dystrophies that can be treated with a inhibiting GDF-ll and/or nodal activities, and even more regimen including the subject myostatin include: Duchenne preferably at least 5, 10, 100 or even 1000 times less. 30 Muscular Dystrophy (DMD), Becker Muscular Dystrophy In certain embodiments, the subject variant Cerberus/Coco (BMD), Emery-Dreifuss Muscular Dystrophy (EDMD), polypeptide is selective for binding and inhibition of myosta Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulo tin, e.g., relative to other BMP proteins such as BMP-4. For humeral Muscular Dystrophy (FSH or FSHD) (Also known instance, the variant Cerberus/Coco polypeptide can be one as Landouzy-Dejerine), Myotonic Dystrophy (MMD) (Also which has a dissociation constant (Kd) for myostatin binding 35 known as Steinert’s Disease), Oculopharyngeal Muscular that is at least 2 times less than its Kd for binding BMP-4, and Dystrophy (OPMD), Distal Muscular Dystrophy (DD), and even more preferably at least 5, 10, 100 or even 1000 times Congenital Muscular Dystrophy (CMD). less. Whether by virture of binding kinetics or biodistribution, Exemplary motor neuron diseases that can be treated with the subject variant Cerberus/Coco polypeptide can also be a regimen including the subject myostatin include: Amyo selected based on relative in vivo potency, such as an inhibitor 40 trophic Lateral Sclerosis (ALS) (Also known as Lou Gehrig’ s that has an EC50 for inhibiting myostatin activity, or a par Disease), Infantile Progressive Spinal Muscular Atrophy ticular physiological consequence (such as promoting muscle (SMA, SMAl or WH) (Also known as SMA Type 1, Werd growth) that is at least 2 times less than its EC50 for inhibiting nig-Hoffman), Intermediate Spinal Muscular Atrophy (SMA BMP-4 activities, and even more preferably at least 5, 10, 100 or SMA2) (Also known as SMA Type 2), Juvenile Spinal or even 1000 times less. 45 Muscular Atrophy (SMA, SMA3 or KW) (Also known as In certain preferred embodiments, the variant Cerberus/ SMA Type 3, Kugelberg-Welander), Spinal Bulbar Muscular Coco polypeptide binding domain binds myostatin with a K d Atrophy (SBMA) (Also known as Kennedy’s Disease and of 1 uM or less, and more preferably a Kd of 100 nM, 10 nM X-Linked SBMA), and Adult Spinal Muscular Atrophy or even 1 nM or less. (SMA). In general, the subject myostatin inhibtor preparations are 50 Exemplary in?ammatory myopathies that can be treated suitable for use in a human patients. In preferred embodi with a regimen including the subj ect myostatin include: Der ments, the subject preparations of variant Cerberus/Coco matomyositis (PM/DM), Polymyositis (PM/DM), and Inclu polypeptides will be substantially free of pyrogenic materials sion Body Myositis (IBM). so as to be suitable for administration to a human patient. Exemplary diseases of the neuromuscular junction that can In other embodiments, the subject variant Cerberus/Coco 55 be treated with a regimen including the subject myostatin polypeptides can be administered to non-human animals, par include: Myasthenia Gravis (MG), Lambert-Eaton Syndrome ticularly other mammals. For example, the compounds of the (LES), and Congenital Myasthenic Syndrome (CMS). present disclosure can be given to chickens, turkeys, livestock Exemplary myopathies due to endocrine abnormalities that animals (such as sheep, pigs, horses, cattle, etc.), companion can be treated with a regimen including the subject myostatin animals (e.g., cats and dogs) or may have utility in aquacul 60 include: Hyperthyroid Myopathy (HYPTM) and Hypothy ture to accelerate growth and improve the protein/ fat ratio. To roid Myopathy (HYPOTM). further illustrate, the subject variant Cerberus polypeptides Exemplary diseases of peripheral nerve that can be treated can be used to stimulate growth or enhance feed ef?ciency of with a regimen including the subject myostatin include: Char animals raised for meat production to improve carcass qual cot-Marie-Tooth Disease (CMT), Dejerine-Sottas Disease ity, or to increase milk production in dairy cattle. 65 (DS), and Friedreich’s Ataxia (FA). Another aspect of the disclosure relates to packaged phar Other exemplary myopathies that can be treated with a maceuticals comprising a pharmaceutical preparation of a regimen including the subject myostatin include: Myotonia US 8,796,199 B2 7 8 Congenita (MC), Paramyotonia Congenita (PC), Central 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more Core Disease (CCD), Nemaline Myopathy (NM), Myotubu of the following: the sequence of amino acids 101-189 of lar Myopathy (MTM or MM), and Periodic Paralysis (PP). human Coco, the sequence of amino acids 95-185 of human Exemplary metabolic diseases of muscle that can be Coco, the sequence of amino acids 95-189 of human Coco, treated with a regimen including the subject myostatin the sequence of amino acids 22-185 of human Coco, or the include: Phosphorylase De?ciency (MPD or PYGM), Acid sequence of amino acids 22-189 of human Coco. Maltase De?ciency (AMD), Phosphofructokinase De? In certain embodiments, the myostatin antagonist protein ciency (PFKM), Debrancher Enzyme De?ciency (DBD), retains at least 50% of the myostatin antagonist activity after Mitochondrial Myopathy (MITO), Camitine De?ciency exposure to human serum for 24 hours at 37° C. The myosta (CD), Camitine Palmityl Transferase De?ciency (CPT), tin antagonist activity may be assessed, for example, in an Phosphoglycerate Kinase De?ciency (PGK), Phosphoglyc A204 cell based assay. erate Mutase De?ciency (PGAM or PGAMM), Lactate In certain embodiments, the myostatin antagonist protein Dehydrogenase De?ciency (LDHA), and Myoadenylate comprises a modi?cation with respect to the amino acid Deaminase De?ciency (MAD). sequence of SEQ ID NO:5 such that cleavage in human serum The subject method can also be used to prevent, ameliorate is reduced or eliminated. The modi?cation with respect to the or reduce the severity of a metabolic disorder, such as in the amino acid sequence of SEQ ID NO:5 may reduce or elimi treatment of obesity or type II diabetes. To further illustrate, nate cleavage within one or both of the following sequences: the subject variant Cerberus/Coco polypeptide preparations PARKRW (SEQ ID NO: 25) or SRRRVK (SEQ ID NO: 26) can be used to decrease body fat proportion in a subject. of human Coco. In still other embodiments, the variant Cerberus/Coco 20 In certain embodiments, the myostatin antagonist protein polypeptide preparations can be used as part of such methods may be a fusion protein including one additional polypeptide as reducing frailty associated with aging. portion that enhances one or more of in vivo stability, in vivo The subject pharmaceutical composition can also be used half life, uptake/administration, tissue localization or distri as myostatin antagonist to treat a number of neuronal system bution, formation of protein complexes, and/or puri?cation. disease conditions, including CNS injuries/disease such as 25 In certain embodiments, the fusion protein includes a portion spinal cord injury and stroke, and PNS injuries/ diseases. of an immunoglobulin heavy chain constant domain. In cer In one aspect, the disclosure provides a myostatin antago tain embodiments, the fusion protein comprises an Fc domain nist protein comprising an amino acid sequence that is at least of an immunoglobulin. In certain embodiments, the myosta 90% identical to the sequence of amino acids 162-241 of tin antagonist protein includes one or more modi?ed amino human Cerberus (SEQ ID NO:2), and wherein said protein is 30 acid residues selected from: a glycosylated amino acid, a substantially serum stable for a period of at least 24 hours. PEGylated amino acid, a famesylated amino acid, an acety In certain embodiments, the myostatin antagonist protein lated amino acid, a biotinylated amino acid, an amino acid comprises an amino acid sequence that is at least 90%, 95%, conjugated to a lipid moiety, and an amino acid conjugated to 96%, 97%, 98%, 99%, or 100% identical to one or more of the an organic derivatizing agent. following: the sequence of amino acids 156-241 of human 35 In certain embodiments, the myostatin antagonist protein Cerberus, the sequence of amino acids 156-267 of human is a fusion protein that further comprises a second myostatin Cerberus, the sequence of amino acids 141-241 of human inhibitor domain, which is a polypeptide af?nity reagent that Cerberus, the sequence of amino acids 141-267 of human selectively binds to myo statin and competes with the binding Cerberus, the sequence of amino acids 119-241 of human of an ALK7 or ALK4 receptor. In certain embodiments, the Cerberus, the sequence of amino acids 41-241 of human 40 af?nity reagent is one or more of the following: (i) an anti Cerberus, the sequence of amino acids 41-267 of human body agent, (ii) a peptide or scaffolded peptide that selec Cerberus, the sequence of amino acids 18-241 of human tively binds to myostatin and competes with the binding of an Cerberus, or the sequence of amino acids 18-267 of human ALK7 or ALK4 receptor, (iii) a myostatin binding domain of Cerberus. ALK7 or ALK4, or (iv) small organic molecule that selec In certain embodiments, the myostatin antagonist protein 45 tively binds to myostatin and competes with the binding of an retains at least 50% of the myostatin antagonist activity after ALK7 or ALK4 receptor. Examples of suitable antibody exposure to human serum for 24 hours at 37° C. The myosta agents include, for example, a recombinant antibody; a tin antagonist activity may be assessed, for example, in an monoclonal antibody; aVH domain; aVL domain; an scFv; an A204 cell based assay. Fab fragment; an Fab' fragment; an F(ab')2; an Fv; or a dis In certain embodiments, the myostatin antagonist protein 50 ul?de linked Fv. In certain embodiments, the antibody agent comprises a modi?cation with respect to the amino acid is a fully human antibody or a humanized chimeric antibody, sequence of SEQ ID NO:2 such that cleavage in human serum or an antigen binding fragment thereof. is reduced or eliminated. The modi?cation with respect to the In another aspect, the disclosure provides a pharmaceutical amino acid sequence of SEQ ID NO:2 may reduce or elimi preparation comprising one or more of the myostatin antago nate cleavage within one or more of the following sequences: 55 nist proteins described herein. the sequence SHCLPAK (SEQ ID NO: 22) of human Cer In another aspect, the disclosure provides a method for berus, the sequence MFRKTP (SEQ ID NO: 23) of human inhibiting myostatin and/ or GDFl 1 and/or Nodal in a patient, Cerberus, or the sequence NQRELP (SEQ ID NO: 24) of the method comprising administering to the patient an effec human Cerberus. tive amount of one or more of the myostatin antagonist pro In another aspect, the disclosure provides a myostatin 60 teins described herein. In certain embodiments, inhibiting antagonist protein, the protein comprising an amino acid myostatin and/or GDFll and/or Nodal in a patient causes a sequence that is at least 90% identical to the sequence of detectable change in the expression of a gene that is regulated amino acids 101-185 of human Coco (SEQ ID NO:5), and by myostatin and/or GDFll and/ or Nodal. wherein said protein is substantially serum stable for a period In another aspect, the disclosure provides a method for of at least 24 hours. 65 increasing muscle mass in a patient, the method comprising In certain embodiments, the myostatin antagonist protein administering to the patient an effective amount of one or comprises an amino acid sequence that that is at least 90%, more of the myostatin antagonist proteins described herein. US 8,796,199 B2 1 0 In another aspect, the disclosure provides a pharmaceutical group consisting of: amino acids 162-241 of SEQ ID NO: 2 preparation substantially free of pyrogenic materials, com and amino acids 101-189 ofSEQ ID NO:5. In certain embodi prising a myostatin antagonist protein including a myostatin ments, the myostatin binding domain has an amino acid binding domain of a Cerberus or Coco polypeptide or variant sequence that is at identical to an amino acid sequence thereof, which myostatin antagonist protein: (a) binds to and selected from the group consisting of: amino acids 162-241 of inhibits the signaling activity of one or more of Nodal, SEQ ID NO: 2 and amino acids 101-189 ofSEQ ID NO:5, or GDF1 1 and/ or myostatin; and (b) does not substantially bind any naturally occurring human allelic variant thereof. In cer to BMP4. tain embodiments, inhibiting myostatin and/or GDF11 in a In certain embodiments, the myostatin antagonist protein patient causes a detectable change in the expression of a gene promotes growth of muscle tissue. that is regulated by myostatin and/ or GDF11. In certain In certain embodiments, the myo statin binding domain has embodiments, the myo statin antagonist does not substantially an amino acid sequence that is at least 90%, 95%, 96%, 97%, bind to BMP4. 98%, 99%, or 100% identical to one or both of the following: amino acids 162-241 of SEQ ID NO: 2, amino acids 101 -1 89 In another aspect, the disclosure provides a method for of SEQ ID NO:5. In certain embodiments, the myostatin increasing skeletal muscle mass in a patient in need thereof, binding domain has an amino acid sequence that is at identical the method comprising administering to the patient an effec to an amino acid sequence selected from the group consisting tive amount of a myostatin antagonist protein including a of: amino acids 162-241 of SEQ ID NO: 2 and amino acids myostatin binding domain of a Cerberus or Coco polypeptide 101-189 of SEQ ID NO:5, or any naturally occurring human or variant thereof, which myostatin antagonist protein binds allelic variant thereof. 20 to and inhibits the signaling activity of one or more of nodal, In certain embodiments, the myostatin antagonist protein GDF11 and/ or myostatin. In certain embodiments, the myo does not include a full-length mature human Cerberus pro statin binding domain has an amino acid sequence that is at tein. least 80% identical to an amino acid sequence selected from In certain embodiments, the myostatin antagonist protein the group consisting of: amino acids 162-241 ofSEQ ID NO: is a fusion protein including one additional polypeptide por 25 2 and amino acids 101-189 of SEQ ID NO:5. In certain tion that enhance one or more of in vivo stability, in vivo half embodiments, the myostatin binding domain has an amino life, uptake/administration, tissue localization or distribution, acid sequence that is at identical to an amino acid sequence formation of protein complexes, and/or puri?cation. In cer selected from the group consisting of: amino acids 162-241 of tain embodiments, the fusion protein includes a portion of an SEQ ID NO: 2 and amino acids 101-189 ofSEQ ID NO:5, or immunoglobulin heavy chain constant domain. In certain 30 any naturally occurring human allelic variant thereof. In cer embodiments, the fusion protein comprises an Fc domain of tain embodiments, the myostatin antagonist does not substan an immuno globulin. In certain embodiments, the myostatin antagonist protein tially bind to BMP4. In another aspect, the disclosure provides use of a myosta includes one or more modi?ed amino acid residues selected tin antagonist protein including a myostatin antagonist pro from: a glycosylated amino acid, a PEGylated amino acid, a 35 famesylated amino acid, an acetylated amino acid, a biotiny tein including a myostatin binding domain of a Cerberus or lated amino acid, an amino acid conjugated to a lipid moiety, Coco polypeptide or variant thereof, which myo statin antago and an amino acid conjugated to an organic derivatizing nist protein binds to and inhibits the signaling activity of one agent. or more of nodal, GDF1 1 and/ or myostatin for the preparation In certain embodiments, the myostatin antagonist protein 40 of a medicament for promoting growth of muscle tissue in a is a fusion protein that further comprises a second myostatin mammal. inhibitor domain, which is a polypeptide a?inity reagent that selectively binds to myo statin and competes with the binding BRIEF DESCRIPTION OF THE DRAWINGS of an ALK7 or ALK4 receptor. In certain embodiments, the af?nity reagent is one or more of the following: (i) an anti 45 FIG. 1 shows a schematic drawing of where Wnt, Nodal body agent, (ii) a peptide or scaffolded peptide that selec and BMP bind to Cerberus. BMP-2 and the highly related tively binds to myostatin and competes with the binding of an BMP-4 competitively bind Cerberus, likely in the same ALK7 or ALK4 receptor, (iii) a myostatin binding domain of region. Other more distantly related or unrelated proteins, ALK7 or ALK4, or (iv) a small organic molecule that selec such as TGF-betal, EGF, and PDGF, do not compete with tively binds to myostatin and competes with the binding of an 50 BMP-4. The N-terminally truncated version of Cerberus still ALK7 or ALK4 receptor. Examples of suitable antibody binds an-l (Xenopus homolog of mouse Nodal). (Adapted agents include, for example, a recombinant antibody; a from Piccolo et al., Nature 397: 707-710, 1999). monoclonal antibody; aVH domain; aVL domain; an scFv; an FIG. 2 Binding of Caronte-Fc to GDP-11. The tracing Fab fragment; an Fab' fragment; an F(ab')2; an Fv; or a dis shows that Caronte-Fc binds to GDP-11 on a BiaCore chip. ul?de linked Fv. In certain embodiments, the antibody agent 55 GDP-11 was immobilized on a BiaCore CM5 chip using is a fully human antibody or a humanized chimeric antibody, standard amine coupling procedure. Trace: Caronte-Fc (200 or an antigen binding fragment thereof. ug/ml; R&D Systems) was injected on the GDP-11 coupled In another aspect, the disclosure provides a method for chip. inhibiting myostatin and/or GDF11 in a patient, the method FIG. 3 A-204 Reporter Gene Assay. The ?gure shows the comprising administering to the patient an effective amount 60 Reporter vector: pGL3 (CAGA)12 (described in Dennler et al, of a myostatin antagonist protein including a myostatin bind 1998, EMBO 17: 3091-3100.) The CAGA12 motif (SEQ ID ing domain of a Cerberus or Coco polypeptide or variant NO: 30) is present in TGF-Beta responsive genes (PAI-l thereof, which myostatin antagonist protein binds to and gene), so this vector is of general use for factors signaling inhibits the signaling activity of one or more of nodal, GDF1 1 through Smad2 and 3. and/ or myostatin. In certain embodiments, the myostatin 65 FIG. 4 Caronte-Fc inhibits GDP-11 signaling in the A-204 binding domain has an amino acid sequence that is at least Reporter Gene Assay. AnActRIIA-Fc (“IIA muG2a”) fusion 90% identical to an amino acid sequence selected from the also inhibits GDP-11 signaling. US 8,796,199 B2 1 1 12 FIG. 5 Caronte-Fc does not inhibit Activin A in the A-204 subject. For instance, the pharmaceutical preparations of the Reporter Gene Assay. AnActRHA-Fc fusion (“IIA muG2a”), present disclosure can be administered in an amount effective as expected, does inhibit ActivinA signaling. to prevent, ameliorate or reduce the severity of a wasting FIG. 6 Cerberus-Fc and Caronte-Fc both inhibit GDP-8 disorder, such as cachexia, anorexia, DMD syndrome, BMD signaling in the A-204 Reporter Gene Assay. syndrome, AIDS wasting syndrome, muscular dystrophies, FIG. 7 Human Cerberus-Fc is degraded in human serum. neuromuscular diseases, motor neuron diseases, diseases of Conditioned medium from cells expressing human Cerberus the neuromuscular junction, and in?ammatory myopathies. Fc was incubated overnight at 37 deg. C. with varying amounts of human serum (percentages of serum added are II. De?nitions shown at top), and resolved by SDS-PAGE. Cerberus was detected by Western blot with a primary antibody: biotiny The terms used in this speci?cation generally have their lated polyclonal anti-cerberus, and a secondary antibody: ordinary meanings in the art, within the context of this dis avidin-HRP. The left lane is molecular weight standards. The closure and in the speci?c context where each term is used. major band, at roughly 70 kD is Cerberus-Fc, which is com Certain terms are discussed below or elsewhere in the speci pletely degraded when incubated with 5% human serum. ?cation, to provide additional guidance to the practitioner in FIG. 8 Human Coco-Fc (murine Fc) inhibits GDP-11 sig describing the compositions and methods of the disclosure naling in a cell based assay. Conditioned medium from cells and how to make and use them. The scope an meaning of any expressing human Coco-ch was tested for effects on A-204 use of a term will be apparent from the speci?c context in reporter gene expression in the presence of GDP-11. which the term is used. 20 “About” and “approximately” shall generally mean an DETAILED DESCRIPTION acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exem I. Overview plary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value Cerberus is expressed in the anterior endomesoderm (Bou 25 or range of values. wmeester et al., Nature 382: 595-601, 1996; Piccolo et al., Alternatively, and particularly in biological systems, the Nature 397: 707-10, 1999; Rodriguez et al., Nature 401: terms “about” and “approximately” may mean values that are 243-51, 1999) during development. Caronte, a chick within an order of magnitude, preferably within 5-fold and ortholog, is involved in left-right asymmetry in the chick more preferably within 2-fold of a given value. Numerical embryo (Rodriguez, supra). Cerberus functions as a multiva 30 quantities given herein are approximate unless stated other lent growth factor antagonist in the extracellular space and wise, meaning that the term “about” or “approximately” can inhibits signaling by BMP-4, nodal, and Wnt (Belo et al., be inferred when not expressly stated. Genesis 26: 265-70, 2000). Mouse Cerberus binds to BMP The methods of the disclosure may include steps of com proteins and nodal via independent sites (Piccolo, supra), paring sequences to each other, including wild-type sequence whereas the Xenopus Cerberus also binds Wnt proteins and 35 to one or more mutants/ sequence variants Such comparisons inhibits their actions (Belo, supra). Cerberus has the unique typically comprise alignments of polymer sequences, e.g., property of inducing ectopic heads in the absence of trunk using sequence alignment programs and/or algorithms that structures (Piccolo, supra). The expression of Cerberus dur are well known in the art (for example, BLAST, FASTA and ing gastrulation is activated by nodal-related signals in endo MEGALIGN, to name a few). The skilled artisan can readily derm and by Spemann-organizer factors (Yamamoto et al., 40 appreciate that, in such alignments, where a mutation con Dev Biol 257: 190-204, 2003). tains a residue insertion or deletion, the sequence alignment Orthologs for Cerberus can be found in Xenopus lropicalis will introduce a “gap” (typically represented by a dash, or and Fugu rubripes, but are missing in invertebrates. In Fugu “A”) in the polymer sequence not containing the inserted or rubripes, there is only one ortholog for Cerberus. All ortholo deleted residue. gous genes for Cerberus have two exons; the ?rst eight amino 45 “Homologous,” in all its grammatical forms and spelling acids of the cystine-knot domain are encoded by the 3' end of variations, refers to the relationship between two proteins that the ?rst exon and the remainder of the motif by the second possess a “common evolutionary origin,” including proteins exon. In some orthologs, a predicted proteolytic cleavage site from superfamilies in the same species of organism, as well as can be found upstream of the beginning of the cystine-knot homologous proteins from different species of organism. domain. 50 Such proteins (and their encoding nucleic acids) have Coco is another member of the Cerberus/Dan family of sequence homology, as re?ected by their sequence similarity, proteins that inhibits Nodal signaling. whether in terms of percent identity or by the presence of In part, the present disclosure provides Coco or Cerberus speci?c residues or motifs and conserved positions. derivatives for inhibiting Nodal, GDP-11 and/or myostatin The term “sequence similarity,” in all its grammatical function. In certain embodiments, the Coco and Cerberus 55 forms, refers to the degree of identity or correspondence derivatives inhibit Nodal, GDF-l 1 and/or myostatin function between nucleic acid or amino acid sequences that may or without substantially compromising BMP (such as BMP-4) may not share a common evolutionary origin. signaling (e.g., does not substantially bind BMP-4 or other However, in common usage and in the instant application, BMPs). The subject Cerberus derivatives may also be used to the term “homologous,” when modi?ed with an adverb such inhibit BMP (such as BMP-4) signaling. 60 as “highly,” may refer to sequence similarity and may or may Exemplary preparations of the subject disclosure include not relate to a common evolutionary origin. Cerberus polypeptide derivatives, including the N-terminal A nucleic acid molecule is “hybridizable” to another truncated versions of Cerberus or Coco. These so-called nucleic acid molecule, such as a cDNA, genomic DNA, or “Cerberus derivatives” or “Coco derivatives” can be used to RNA, when a single stranded form of the nucleic acid mol reduce the severity of a pathologic condition, which is char 65 ecule can anneal to the other micleic acid molecule under the acterized, at least in part, by an abnormal amount, develop appropriate conditions of temperature and solution ionic ment or metabolic activity of muscle or adipose tissue in a strength (see Sambrook et al. Molecular Cloning: A Labora US 8,796,199 B2 13 14 Zory Manual, Second Edition (1989) Cold Spring Harbor acid is at least about 10 nucleotides; preferably at least about Laboratory Press, Cold Spring Harbor, N.Y.). The conditions 15 nucleotides; and more preferably the length is at least of temperature and ionic strength determine the “stringency” about 20 nucleotides. of the hybridization. For preliminary screening for homolo Unless speci?ed, the term “standard hybridization condi gous nucleic acids, low stringency hybridization conditions, tions” refers to a Tm of about 55° C., and utilizes conditions as corresponding to a Tm (melting temperature) of 55° C., can be set forth above. In a preferred embodiment, the Tm is 60° C.; used, e.g., 5> Murine Cerberus protein (NCBI RefSeq ID NP_O340l7) (SEQ ID NO: 1): lMHLLLVQLLV LLPLGKADLC VDGCQSQGSL SFPLLERGRR DLHVANHEEA EDKPDLFVAV 61PHLMGTSLAG EGQRQRGKML SRLGRFWKKP ETEFYPPRDV ESDHVSSGMQ AVTQPADGRK lZlVERSPLQEEA KRFWHRFMFR KGPAFQGVIL PIKSHEVHWE TCRTVPFNQT IAHEDCQKVV l8lVQNNLCFGKC SSIRFPGEGA DAHSFCSHCS PTKFTTVHLM LNCTSPTPVV KMVMQVEECQ 24lCMVKTERGEE RLLLAGSQGS FIPGLPASKT NP US 8,796,199 B2 17 18 —continued 1621 gaaatcttct actcgtatgc ttttgaatct tctagcaagt taggtttcta tgtttgggct 1681 tcttcctatt gtctaagagt atgtgtgaca aattcaacct gacaaatacc tcaatggcaa 1741 attctgaccc tg NCBI RefSeq ID NM_005454.1 (human Cerberus mRNA). (SEQ ID NO: 1 atgcatctcc tcttatttca gctgctggta ctcctgcctc taggaaagac cacacggcac 61 caggatggcc gccagaatca gagttctctt tcccccgtac tcctgccaag gaatcaaaga 121 gagcttccca caggcaacca tgaggaagct gaggagaagc cagatctgtt tgtcgcagtg 181 ccacaccttg tagccaccag ccctgcaggg gaaggccaga ggcagagaga gaagatgctg 241 tccagatttg gcaggttctg gaagaagcct gagagagaaa tgcatccatc cagggactca 301 gatagtgagc ccttcccacc tgggacccag tccctcatcc agccgataga tggaatgaaa 361 atggagaaat ctcctcttcg ggaagaagcc aagaaattct ggcaccactt catgttcaga 421 aaaactccgg cttctcaggg ggtcatcttg cccatcaaaa gccatgaagt acattgggag 481 acctgcagga cagtgccctt cagccagact ataacccacg aaggctgtga aaaagtagtt 541 gttcagaaca acctttgctt tgggaaatgc gggtctgttc attttcctgg agccgcgcag 601 cactcccata cctcctgctc tcactgtttg cctgccaagt tcaccacgat gcacttgcca 661 ctgaactgca ctgaactttc ctccgtgatc aaggtggtga tgctggtgga ggagtgccag 721 tgcaaggtga agacggagca tgaagatgga cacatcctac atgctggctc ccaggattcc 781 tttatcccag gagtttcagc ttga It is also expected that Cerberus related proteins also exist domain. A Cerberus derivative or variant sequence may or in other species, including family members in Xenopus, and may not lack the N-terminal BMP binding domain. A variety Drosophila, C. elegans, zebra?sh, as well as in all manmnals, of allelic variants of human Cerberus are known, including 35 for example, rats, mice and humans. “Cerberus or Cerberus A65G (alanine 65 to glycine), V1791 and L221V. like proteins” also includes variants of the Cerberus proteins, “Coco or Coco-like protein” refers to mammalian Coco such as allelic variants or variants induced by mutagenesis or proteins and related homologs, such as the human Coco pro deletions, and fragments of Cerberus proteins which variants tein of GenBank Accession 22749329, and other proteins and fragments retain myostatin binding activity. “Cerberus 40 which share sequence homology to the highly conserved like” proteins is also used to signify the family of proteins cysteine pattern of the C-terminal portion of the mammalian sharing structural and/or functional similarity, including Coco proteins. An exemplary amino acid sequences for those proteins which are described further herein. Such pro human Coco protein is (SEQ ID NO: 5) lMLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSW lZlRNHLCFGHCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLI l8lEGCHCSPKA teins may have amino acid sequences sharing signi?cant Amino acids 1-21 of SEQ ID NO:5 correspond to a signal sequence identity (e.g., at least about 50%, 60%, 70%, 80%, peptide that may be replaced with an alternative leader 90%, 95%, 99% or more) with the human or mouse Cerberus sequence. A mature secreted Coco polypeptide is expected to 60 correspond to amino acids 22-189 of SEQ ID N015, although proteins, over the full-length, or at least within the myostatin imprecisions in the signal peptide processing enzymes may binding domain of the human or mouse Cerberus. Cerberus lead to alternative or additional cleavage at positions ranging like proteins also include proteins that have amino acid from one to ?ve amino acids towards the amino terminus or sequences that are encoded by nucleic acid sequences that the carboxy terminus from the glycine at position 22. As hybridize under stringent conditions with the coding 65 disclosed herein, a tPA leader sequence or other heterologous sequences for human or mouse Cerberus, particularly that leader sequence may be used in place of the native leader portion of the coding sequence for the myostatin binding sequence. Proposed leader sequences are as follows: US 8,796,l99 B2 19 20 (SEQ ID NO: 14) (i) Honey bee mellitin (HBML): (SEQ ID NO: 15) (ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP The human Coco coding sequence is disclosed in GenBank Accession 22749328 (incorporated herein by reference). (SEQ ID NO: 6) 1 agtccggaca gacagacagg cagacagacg cacggacaag cagatgctcc ttggccagct 61 atccactctt ctgtgcctgc ttagcggggc cctgcctaca ggctcaggga ggcctgaacc 121 ccagtctcct cgacctcagt cctgggctgc agccaatcag acctgggctc tgggcccagg 181 ggccctgccc ccactggtgc cagcttctgc ccttgggagc tggaaggcct tcttgggcct 241 gcagaaagcc aggcagctgg ggatgggcag gctgcagcgt gggcaagacg agthQCtgc 301 tgtgactctg ccgctgaacc ctcaggaagt gatccagggg atgtgtaagg ctgtgccctt 361 cgttcaggtg ttctcccggc ccggctgctc agccatacgc ctccgaaatc atctgtgctt 421 tggtcattgc tcctctctct acatccctgg ctcggacccc accccactag tcctgtgcaa 481 cagctgtatg cctgctcgca agcgttgggc acccgtggtc ctgtggtgtc tcactggcag 541 ctcagcctcc cgtcgacggg tgaagatatc caccatgctg atcgaggggt gtcactgcag 601 cccaaaagca tgaactgagc atcgtggatg ggtgcacgga gacacgcacc ttggagaaat 661 gaggggagat ggaccaagaa agacgtggac ctggatgatg tactctgggt caagagacca 721 gggatgcagg gttaggcaga caggtcccca gagtcctcac cctgctcccc agacagtaga 781 cacagtgccc gtcctggagt tgcaccactg atagtcacag cacacaatga ttgacaactc 841 actttttttt ttttttttga gatggagtct cgctctgtcg cccaggctgg agtgcagtgg 901 cgcaatctca gctcactgca agctccacct cccgggttta tgccattctc ctgtctcagc 961 ctcccgagta gctgggacta caggcacccg ccaacacgcc cggctaattt ttcgtatttt 1021 tagtaaagac agggtttcac cgtgttagcc aggatggtct ctatctcctg acctcgtgat 1081 ctgcctgcct tggccttatt attttttttt tttaaggaca gagtctctct ctgtcaccca 1141 ggctggagtg caatggcgcg atcttggctc actgtaactt ccacttgcca ggctcaagca 1201 gttctcctgc ctcagcctcc tgagtagctg ggactacagg cacccgccac catgcccagc 1261 taatttttgt atttttagta gagacagagt ttcaccatat tagcctggct ggtctcaaac 1321 tcctggcctc aggtgatctg cccacctcgg cctcccaaag tgctgggatc aaatccactg 1381 ttaatcatta ggctgaactg tctcttatag aatgaggtca aagacactcc cagttgcagg 1441 gagggtagat ggccccaccc agaccgagag acacagtgat gacctcagcc tagggacacc 1501 aaaaaaaaaa aaaaaaaaaa cccaaaccaa aaacgcaaac caaagcaggc aggcagacag 1561 ctgctggggg aaatcctggg gtccttgaga cagaggcagg accctcgtgt tcccagctgc 1621 ctcttgcctt gatagtggtg ctgtgtccct ctcagacccc ccacctgagt ctccacagag 1681 ccccacgcct ggcatggcat tccacagaaa ccataaaggt tggctgagtc c It is also expected that Coco-related proteins also exist in larity, including those proteins Which are described further other species, including family members in Xenopus, and herein. Such proteins may have amino acid sequences sharing Drosophila, C. elegans, zebra?sh, as well as in all manmnals, 60 signi?cant sequence identity (e.g., at least about 50%, 60%, for example, rats, mice and non-human primates. “Coco or 70%, 80%, 90%, 95%, 99% or more) With the human Coco Coco-like proteins” also includes variants of the naturally protein, over the full-length, or at least Within the myostatin occurring Coco proteins, such as allelic variants or variants binding domain of the human Coco. Coco-like proteins also induced by mutagenesis or deletions, and fragments of Coco include proteins that have amino acid sequences that are proteins Which variants and fragments retain myostatin bind 65 encoded by nucleic acid sequences that hybridize under strin ing activity. “Coco-like” proteins is also used to signify the gent conditions With the coding sequences for human Coco, family of proteins sharing structural and/ or functional simi particularly that portion of the coding sequence for the myo