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(12) United States Patent (10) Patent No.: US 7,833,971 B2 Grinberg Et Al

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(12) United States Patent (10) Patent No.: US 7,833,971 B2 Grinberg Et Al US007833971B2 (12) United States Patent (10) Patent No.: US 7,833,971 B2 Grinberg et al. (45) Date of Patent: Nov. 16, 2010 (54) USES OF CERBERUS, COCO AND WO WO-03/O12082 2, 2003 DERVATIVES THEREOF WO WO-03/05.5911 T 2003 WO WO-O3,O72714 9, 2003 (75) Inventors: Asya Grinberg, Boston, MA (US); John WO WO-03/106657 12/2003 Knopf, Carlisle, MA (US); Ravindra WO WO-2004/074460 9, 2004 Kumar, Shrewsbury, MA (US); Jasbir WO WO-2005/003 158 1, 2005 Seehra, Lexington, MA (US) WO WO-2005/115439 A2 12/2005 (73) Assignee: Acceleron Pharma Inc., Cambridge, OTHER PUBLICATIONS MA (US) Avsian-Kretchmer et al., “Comparative geneomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic (*) Notice: Subject to any disclaimer, the term of this protein antagonists.” Molecular Endocrinology 18(1): 1-12 (2004). patent is extended or adjusted under 35 Belo et al., “Cerberus-like is a secreted factor with nerualizing activ U.S.C. 154(b) by 0 days. ity expressed in the anterior primitive endoderm of the mouse gas trula.” Mechanisims of Development 68:45-57 (1997). (21) Appl. No.: 12/001,494 Biben et al., “Murine Cerberus Homologue mCer-1: A canadidate anterior patterning molecule.” Developmental Biology 194:135-151 (22) Filed: Dec. 10, 2007 (1998). Bouwmeester et al., “Cerberus is a head-inducing secreted factor (65) Prior Publication Data expressed in the anterior endoderm of Spemann's organizer. Nature US 2009/OO82270 A1 Mar. 26, 2009 382:595-601 (1996). Esteban et al., “The novel Cer-like protein Caronte mediates the extablishment of embryonic left-right asymmetry.” Nature 401:243 Related U.S. Application Data 251 (1999). (60) Provisional application No. 60/873,933, filed on Dec. Katoh et al., “Identification and characterization of human 8, 2006. CKTSFIB2 and CKTSFIB3 genes in silico.” Oncology Reports 12:423-427 (2004). Kuroda et al., “Neural Induction in Xenopus. requirement for (51) Int. Cl. extodermal and endomesodermal signals via chordin, noggin, C7K I4/0 (2006.01) B-catenin and cerberus.” PLoS Biology 2(5):0623-0634 (2004). (52) U.S. Cl. ........................................... 514/2:530/350 Lahet al., “Human cerberus related gene CERI maps to chromosome (58) Field of Classification Search ....................... None 9.” Genomics 55:364-366 (1999). See application file for complete search history. Marques et al., “The activity of the nodal antagonist Cerl-2 in the mouse node is required for correct LR body axis.” Genes & Devel (56) References Cited opment 18:2342-2347 (2004). U.S. PATENT DOCUMENTS Pearce et al., “A mouse cerberus/dan-related gene family.” Develop mental Biology 209:98-110 (1999). 5,935,852 A 8, 1999 Follettie et al. Piccolo et al., “The head inducer Cerberus is a multifunctional 6,133,232 A 10, 2000 De Robertis et al. antagonist of Nodal, BMP and Wnt signals.” Nature 397:707-710 6,610,510 B1 8, 2003 Valenzuela et al. (1999). 7,316,998 B2 1/2008 Knopfet al. Silva et al., “Endogenous Cerberus activity is required for anterior 2002fO164682 A1 11/2002 Follettie et al. head specification in Xenopus.” Development 130(20):4943-4953 2003/0134790 A1 7/2003 Langenfeld (2003). 2003. O194704 A1 10/2003 Penn et al. Stanley et al., “Murine cerberus homologue Cer 1 maps to chromo 2003. O199042 A1 10/2003 Valenzuela et al. some 4.” Genomics 49:337-338 (1998). 2004.0005560 A1 1/2004 Isogai et al. Motoko Yanagita. BMP antagonists: Their roles in development and 2004/O181033 A1 9, 2004 Han et al. involvement in pathophysiology. Cytokine and Growth Factor 2005, 0186663 A1 8, 2005 Davies et al. Reviews. vol. 16, No. 3, pp. 309-319. 2005. 2006/0105376 A1 5/2006 Isogai et al. Brecher, A.S., et al., "Acetaldehyde Inhibits Chymotrypsin and 2008, OO32304 A1 2/2008 Isogai et al. Serum Anti-Chymotrypsin Activity.” J. Investig Med., 46(4): 146-152 (1998). Abstract only. FOREIGN PATENT DOCUMENTS Livingston, S.F., et al., “The Significance of Chymotrypsin-Inhibitor EP 1347046 9, 2003 Levels in the Serum of Patients with Carcinoma of the Breast.” WO WO-97/.48275 12/1997 Cancer Research, 17(9):857-861 (1957). WO WO-98,34951 8, 1998 WO WO-98/49296 11, 1998 Primary Examiner Karen Cochrane Carlson WO WO-99/O1553 1, 1999 (74) Attorney, Agent, or Firm Ropes & Gray LLP WO WO-99/40181 8, 1999 WO WO-OO,55193 9, 2000 (57) ABSTRACT WO WO-02/10214 2, 2002 WO WO-O2/32929 4/2002 The disclosure relates to Cerberus/Coco polypeptides or vari WO WO-O2/O54940 T 2002 WO WO-O2/O77204 10, 2002 ants thereof for use in treating a variety of disorders associ WO WO-O2/O78516 10, 2002 ated with myostatin, nodal and GDF-11. WO WO-O2/O90992 11, 2002 WO WO-03/055443 1, 2003 6 Claims, 8 Drawing Sheets U.S. Patent Nov. 16, 2010 Sheet 1 of 8 US 7,833,971 B2 Figure 1 U.S. Patent Nov. 16, 2010 Sheet 2 of 8 US 7,833,971 B2 RU 2000 1700 1400 1100 800 500 200 -100 -100 O 100 200 300 400 500 600 TIME S Figure 2 U.S. Patent Nov. 16, 2010 Sheet 3 of 8 US 7,833,971 B2 SYNTHETIC poly(A) SGNA / TRANSCRIPTIONAL PAUSE SITE (FOR 3ACKGROUND - - REDUCTION, / 3V1 Amp' f of pG-3BASIC VECTOR ws.xx-x-r-w f : 20052004 Ban H N.Ya P. poly(A)SIGNALSy40 ATE K, -3. (FOR CREPORTER, Xball 742 Hipg 1902 Figure 3 U.S. Patent Nov. 16, 2010 Sheet 4 of 8 US 7,833,971 B2 U.S. Patent Nov. 16, 2010 Sheet 5 of 8 US 7,833,971 B2 8 8.: 3. 38: U.S. Patent Nov. 16, 2010 Sheet 6 of 8 US 7,833,971 B2 U.S. Patent Nov. 16, 2010 Sheet 7 of 8 US 7,833,971 B2 S.Po g human serum added to CM (37 deg. Covernight) O 1 1 25 5 U.S. Patent Nov. 16, 2010 Sheet 8 of 8 US 7,833,971 B2 Inhibition of GDF-11 by human Coco mfc (Conditioned Medium) 0.6 O.5 0.4 0.3 -0 - GDF-11 + COCO CM 0.2 0.1 O O O1 0.1 1 10 100 Media Dilutions Figure 8 US 7,833,971 B2 1. 2 USES OF CERBERUS, COCO AND statin reduced-function mutation in a human child is associ DERVATIVES THEREOF ated with gross muscle hypertrophy and a family history of exceptional strength (Schuelke et al. 2004 Jun. 24; 350(26): CROSS-REFERENCE TO RELATED 2682-8). An antibody against myostatin is reported to have APPLICATIONS 5 beneficial effects in animal models of muscle disorders, including amyotrophic lateral Sclerosis (Holzbauer et al. This application claims the benefit of the filing date of U.S. Neurobiol Dis. 2006 September; 23(3):697–707). Application No. 60/873,933, filed Dec. 8, 2006, which is Additionally, it should be noted that the serum and intra incorporated by reference in its entirety. muscular concentrations of immunoreactive myostatin are 10 increased in HIV-infected men with muscle wasting com BACKGROUND OF THE INVENTION pared with healthy men, and correlate inversely with the fat-free mass index. These data Support the hypothesis that Transforming growth factor-B 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 15 men (Nestor et al., Supra). Mullerian-inhibiting substance (MIS) (for review, see Mas In view of the above findings, 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-B family exert a wide range 20 in Autoimmune Deficiency Syndrome (AIDS), Multiple of biological effects on a large variety of cell types. Many 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 specification; 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 25 lation of myostatin gene expression. system. Activities of the TGF-B 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) 30 human proteins, Cerberus and Coco, that belong to a group of antagonists/repressors. These BMP repressors represent a 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 35 myostatin/GDF11 binding and antagonist activity can be groups of proteins based on structural analysis, especially the separated from the BMP4/2 binding and antagonist activity.
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