Antibodies to myostatin   

This application is a divisional of U.S. application Ser. No. 11/410,886 filed Apr. 24, 2006, which claims the benefit of the filing date of U.S. Provisional application No. 60/674,933, filed Apr. 25, 2005. The above-referenced applications are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

A growing body of evidence indicates that myostatin (mstn, Growth and Differentiation Factor-8, or GDF-8) negatively regulates skeletal muscle growth. For example, a myostatin null mutation in a child has been associated with dramatic muscle hypertrophy without any obvious abnormalities (Schuelke et al. (2004) Myostatin Mutation Associated with Gross Muscle Hypertrophy in a Child. New Engl. J. Med. 350:2682-8). A negative correlation between muscle myostatin protein levels and skeletal muscle mass has also been demonstrated (Schulte, J. N. and Yarasheski, K. E. (2001). Effects of resistance training on the rate of muscle protein synthesis in frail elderly people. Int. J. Sport Nutr. Exerc. Metab. 11 Suppl:S111-820; Walker K S et al. (2004) Resistance training alters plasma myostatin but not IGF-1 in healthy men. Med Sci Sports Exerc. 36(5):787-93.). For example, there is an increased expression of muscle myostatin levels with age and increased myostatin expression has also been shown to contribute to muscle wasting in HIV-infected patients (Gonzalez-cadavid et al. (1998) Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting. PNAS 95:14938-4321). In addition, elevated myostatin levels are found in elderly populations. (Yarasheski K E et al., (2002) Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men with muscle wasting. J. Nutr. Health Aging. 6(5):343-8). Myostatin also influences bone mass as myostatin-deficient mice have increased bone mineral density (Hamrick et al., (2003) Bone Architecture and Disc Degeneration in the Lumbar Spine of Mice Lacking GDF-8 (Myostatin). J. Orthopaedic Res. 21: 1025-1032 (and references therein).

Antibodies to circulating myostatin have been shown to cause increased muscle mass and improved glucose homeostasis in murine models of type 2 diabetes mellitus. Inhibition of myostatin by ip injection of a neutralizing antibody increases skeletal muscle mass, lowers fasting blood glucose and improves glucose sensitivity in obese diabetic mice (Li X. et al. (2002) Inhibition of myostatin increases muscle mass and improves glucose sensitivity in obese, diabetic mice. Poster #224, in Keystone Symposia: Diabetes Mellitus: Molecular Mechanisms, Genetics and New Therapies). In addition, Ay/a mice are known to develop insulin resistance and are used as a model for type 2 diabetes. When Ay/a mice are made devoid of myostatin by deletion of the myostatin locus, they have normal fed glucose and insulin levels, and dramatically lower glucose levels following an exogenous glucose load relative to normal Ay/amice (McPherron et al. (2002) J. Clin. Invest. 109:595-601).

Considering the detrimental muscle, bone and metabolic defects associated with myostatin, there is an urgent need for antibodies as therapeutics that are specific for myostatin and which prevent or treat conditions by reducing myostatin activity, as well as antibodies as diagnostics to identify individuals that are in need of treatment to reduce myostatin activity.

SUMMARY

The invention provides anti-myostatin antibodies, nucleic acids encoding them, vectors and host cells for producing the antibodies, compositions and kits comprising the antibodies and methods of making and using the antibodies. Various embodiments of the invention described in the following numbered paragraphs include, but are not limited to those. 1. A human, chimeric or humanized monoclonal antibody or an antigen-binding portion thereof that specifically binds to myostatin. 2. The monoclonal antibody or antigen-binding portion according to paragraph 1 wherein the myostatin is human myostatin. 3. A monoclonal antibody or antigen-binding portion according to paragraph 1, wherein said antibody or portion selectively binds myostatin over GDF11 by at least 50-fold. 4. A monoclonal antibody or antigen-binding portion according to paragraph 3, wherein said antibody or portion inhibits myostatin binding to an activin type I or type II receptor. 5. A monoclonal antibody or antigen-binding portion according to paragraph 1, wherein the antibody or portion thereof has at least one property selected from the group consisting of: (a) competes for binding to myostatin with an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (b) binds to the same epitope of myostatin as an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (c) binds to myostatin with substantially the same KD as an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—1; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; and (d) binds to myostatin with substantially the same off rate as an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—1; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V. 6. A monoclonal antibody or antigen-binding portion thereof comprising: (a) a CDR set, CDR1, CDR2, and CDR3, that sequentially together are at least 90% identical in amino acid sequence to heavy chain CDRs, CDR1, CDR2, and CDR3, sequentially together, that are included in the amino acid sequence set forth in any one of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85 and 118; (b) a CDR set, CDR1, CDR2, and CDR3, that sequentially together are at least 90% identical in amino acid sequence to light chain CDRs, CDR1, CDR2, and CDR3, sequentially together, that are included in the amino acid sequence set forth in any one of SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 83, 87 and 120; or (c) a first CDR set of (a) and a second CDR set of (b). 7. A monoclonal antibody or antigen-binding portion thereof according to paragraph 1, wherein said antibody comprises heavy chain CDRs CDR1, CDR2, and CDR3, that sequentially together are at least 90% identical in amino acid sequence to heavy chain CDRs, CDR1, CDR2, and CDR3, sequentially together, that are included in the amino acid sequence set forth in any one of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85 and 118. 8. A monoclonal antibody or antigen-binding portion thereof according to paragraph 1, wherein said antibody comprises light chain CDRs CDR1, CDR2, and CDR3, that sequentially together are at least 90% identical in amino acid sequence to light chain CDRs, CDR1, CDR2, and CDR3, sequentially together, that are included in the amino acid sequence set forth in any one of SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 83, 87 and 120. 9. A monoclonal antibody or antigen-binding portion according to paragraph 1, wherein said antibody or antigen-binding portion comprises the heavy chain CDR1, CDR2 and CDR3 sequences found in any one of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85 or 118. 10. A monoclonal antibody or antigen-binding portion according to paragraph 1, wherein said antibody or antigen-binding portion comprises the light chain CDR1, CDR2 and CDR3 sequences found in any one of SEQ ID NOs: 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87 or 120. 11. A monoclonal antibody or antigen-binding portion according to paragraph 1, wherein said antibody or portion comprises a heavy chain that utilizes a human VH 1-02 gene, a human VH 3-21 gene or a human VH 3-23 gene. 12. A monoclonal antibody or an antigen-binding portion according to paragraph 1, wherein said antibody or portion comprises a light chain that utilizes a human VK L2 gene, a human VK A3 gene or a human VK A30 gene. 13. A monoclonal antibody according to paragraph 1 comprising a VH domain at least 90% identical in amino acid sequence to the VH domain in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 or 115. 14. A monoclonal antibody according to paragraph 1 comprising a VL domain at least 90% identical in amino acid sequence to the VL domain in any one of SEQ ID NOs:4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 or 117. 15. A monoclonal antibody or an antigen-binding portion according to paragraph 1 that specifically binds myostatin, wherein: (a) the heavy chain comprises the heavy chain CDR1, CDR2 and CDR3 amino acid sequences of an antibody selected from the group consisting of: 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (b) the light chain comprises the light chain CDR1, CDR2 and CDR3 amino acid sequences of an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; or (c) the antibody comprises a heavy chain of (a) and a light chain of (b). 16. A monoclonal antibody according to paragraph 12 further comprising a VL domain at least 90% identical in amino acid sequence to the variable domain in any one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 or 117. 17. A monoclonal antibody selected from the group consisting of: (a) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4. (b) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 8; (c) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12; (d) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 16; (e) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 20; (f) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24; (g) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28; (h) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 30 and SEQ ID NO: 32; (i) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 34 and SEQ ID NO: 36; (j) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 40; (k) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 42 and SEQ ID NO: 44; and (l) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 115 and SEQ ID NO: 117. 18. A monoclonal antibody or antigen-binding portion thereof selected from the group consisting of: (a) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4. (b) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 8; (c) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12; (d) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 16; (e) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 20; (f) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24; (g) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28; (h) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 30 and SEQ ID NO: 32; (i) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 34 and SEQ ID NO: 36; (j) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 40; (k) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 42 and SEQ ID NO: 44; and (l) an antibody or antigen-binding portion comprising the variable domain amino acid sequences set forth in SEQ ID NO: 115 and SEQ ID NO: 117. 19. A monoclonal antibody or antigen-binding portion thereof comprising a first CDR sequence set comprising a first CDR1, first CDR2 and first CDR3 and a second CDR sequence set comprising a second CDR1, second CDR2 and second CDR3, wherein said first CDR set and said second CDR set each sequentially together have at least 90% identity to the CDR1, CDR2 and CDR3 sequences, sequentially together, of: (a) SEQ ID NO: 2 and SEQ ID NO: 4, respectively; (b) SEQ ID NO: 6 and SEQ ID NO: 8, respectively; (c) SEQ ID NO:10 and SEQ ID NO:12, respectively; (d) SEQ ID NO:14 and SEQ ID NO:16, respectively; (e) SEQ ID NO:18 and SEQ ID NO:20, respectively; (f) SEQ ID NO:22 and SEQ ID NO:24, respectively; (g) SEQ ID NO:26 and SEQ ID NO:28, respectively; (h) SEQ ID NO:30 and SEQ ID NO:32, respectively; (i) SEQ ID NO:34 and SEQ ID NO:36, respectively; (j) SEQ ID NO:38 and SEQ ID NO:40, respectively; (k) SEQ ID NO:42 and SEQ ID NO:44, respectively; and (l) SEQ ID NO:115 and SEQ ID NO:117, respectively. 20. A monoclonal antibody or antigen-binding portion thereof comprising a first CDR sequence set comprising a first CDR1, first CDR2 and first CDR3 and a second CDR sequence set comprising a second CDR1, second CDR2 and second CDR3, wherein said first CDR set and said second CDR set are each the CDR1, CDR2 and CDR3 sequences, sequentially together, of a) SEQ ID NO: 2 and SEQ ID NO: 4, respectively; (b) SEQ ID NO: 6 and SEQ ID NO: 8, respectively; (c) SEQ ID NO:10 and SEQ ID NO:12, respectively; (d) SEQ ID NO:14 and SEQ ID NO:16, respectively; (e) SEQ ID NO:18 and SEQ ID NO:20, respectively; (f) SEQ ID NO:22 and SEQ ID NO:24, respectively; (g) SEQ ID NO:26 and SEQ ID NO:28, respectively; (h) SEQ ID NO:30 and SEQ ID NO:32, respectively; (i) SEQ ID NO:34 and SEQ ID NO:36, respectively; (k) SEQ ID NO:38 and SEQ ID NO:40, respectively; (l) SEQ ID NO:42 and SEQ ID NO:44, respectively; and (m) SEQ ID NO:115 and SEQ ID NO:117, respectively. 21. A monoclonal antibody that specifically binds myostatin comprising the heavy chain amino acid sequence set forth in SEQ ID NO:115 and the light chain amino acid sequence set forth in SEQ ID NO:117. 22. A monoclonal antibody or an antigen-binding portion thereof comprising the variable regions contained in SEQ ID NO:115 and SEQ ID NO:117. 23. A monoclonal antibody or an antigen-binding portion thereof, that specifically binds myostatin comprising CDRs CDR1, CDR2, and CDR3 contained in SEQ ID NO:115 and SEQ ID NO:117. 24. A monoclonal antibody or an antigen-binding portion thereof said monoclonal antibody or antigen-binding portion binds to peptide 1 and peptide 5 portions of myostatin, wherein peptide 1 comprises the amino acid sequence of SEQ ID NO: 103 and peptide 5 comprises the amino acid sequence of SEQ ID NO: 107. 25. An antibody produced by a cell having ATCC Deposit Designation Number selected from the group consisting of PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576. 26. A pharmaceutical composition comprising an antibody or an antigen-binding portion according to any one of paragraphs 1 to 25 and a pharmaceutically acceptable carrier. 27. A pharmaceutical composition according to paragraph 26, further comprising at least one therapeutic agent. 28. A method comprising the step of administering to said subject an antibody or an antigen-binding portion according to any one of paragraphs 1 to 25 or the pharmaceutical composition according to paragraph 26, wherein said antibody, antigen-binding portion or pharmaceutical composition inhibits myostatin activity, wherein said subject is in need of increasing muscle mass, promoting skeletal muscle development, treating a muscle wasting disorder or enhancing skeletal muscle growth. 29. An isolated cell line that produces an antibody or an antigen-binding portion according to any one of paragraphs 1 to 25 or the heavy chain or light chain of said antibody or said antigen-binding portion. 30. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain or an antigen-binding portion thereof or the light chain or an antigen-binding portion thereof of an antibody according to any one of paragraphs 1 to 25. 31. A vector comprising the nucleic acid molecule according to paragraph 30 wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule. 32. A host cell comprising the vector according to paragraph 31 or the nucleic acid molecule according to paragraph 30. 33. A method for producing an anti-myostatin antibody or an antigen-binding portion thereof, comprising culturing the host cell according to paragraph 32 or the cell line according to paragraph 29 under suitable conditions and recovering said antibody or antigen-binding portion. 34. A non-human transgenic organism carrying the nucleic acid according to paragraph 30 either chromosomally or extrachromosomally, wherein the non-human transgenic organism expresses said nucleic acid. 35. A method for isolating an antibody or an antigen-binding portion thereof that specifically binds to myostatin, comprising the step of isolating the antibody from the non-human transgenic organism according to paragraph 34. 36. A method for treating a subject in need thereof with an antibody or an antigen-binding portion thereof that specifically binds to myostatin comprising the steps of: (a) administering to said subject an effective amount of an isolated nucleic acid molecule according to paragraph 30; and (b) expressing said nucleic acid molecule. 37. A method for producing a human monoclonal antibody that specifically binds to myostatin, comprising the steps of: (a) immunizing a non-human transgenic animal that is capable of producing human antibodies with myostatin, an immunogenic portion of myostatin, or a cell or tissue expressing myostatin; (b) allowing the non-human transgenic animal to mount an immune response to myostatin; (c) isolating B lymphocytes from the non-human transgenic animal; and (d) isolating a monoclonal antibody that specifically binds to myostatin from said isolated B lymphocytes. 38. An isolated antibody produced by the method according to paragraph 37. 39. A method for inhibiting the binding of myostatin to cells expressing an activin Type II or IIB receptor comprising contacting the myostatin with an antibody or antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 40. A method for increasing myoblast proliferation comprising contacting a composition comprising myoblasts and myostatin with an antibody or antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 41. A method comprising administering to a subject an antibody or an antigen-binding portion thereof according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity, wherein said subject is in need of improving glucose homeostasis, decreasing fat mass, increasing insulin sensitivity, improving kidney function, decreasing fat accumulation, treating, preventing or inhibiting a disease or condition characterized by bone loss, said disease or condition including osteoporosis, osteopenia, osteoarthritis and bone fractures, treating metabolic syndrome, or counteracting muscle wasting from sustained administration of a glucocorticoid or a steroid hormone during the time that said subject is undergoing treatment with a glucocorticoid or a steroid hormone. 42. A method for reducing myostatin activity in a subject in need thereof comprising the step of administering to said subject a monoclonal antibody or an antigen-binding portion thereof according to any one of paragraphs 1 to 25, wherein said monoclonal antibody or antigen-binding portion inhibits myostatin activity. 43. A method for reversing age-related decline in muscle mass in a subject in need thereof comprising the step of administering to said subject an antibody or an antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 44. A method for increasing myoblast proliferation and differentiation in a subject in need thereof comprising the step of administering to said subject an antibody or antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 45. A method for reducing myostatin-induced activin type IIA or IIB membrane receptor mediated cell signalling in a subject in need thereof comprising the step of administering to said subject an antibody or an antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 46. A method for decreasing myostatin-mediated activation of an activin type I membrane receptor in a subject in need thereof comprising the step of administering to said subject an antibody or an antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 47. A method for reducing myostatin-mediated phosphorylation of one or more R-smad proteins selected from the group consisting of: Smad 2 and Smad 3 in a subject in need thereof comprising the step of administering to said subject an antibody or antigen-binding portion according to any one of paragraphs 1 to 18, wherein said antibody or antigen-binding portion inhibits myostatin activity. 48. A method for increasing expression of a gene selected from the group consisting of: myoD, myf5 and myogenin, in a subject in need thereof comprising the step of administering to said subject an antibody or antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 49. A method for promoting muscle growth, weight gain or aiding in the prevention of frailty in cattle, swine, sheep, chickens, turkeys, horses, fish, dogs and cats in need thereof comprising the step of administering to said subject an antibody or antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 50. A monoclonal antibody or antigen binding portion thereof selected from the group consisting of: (a) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO: 45 and SEQ ID NO:47; (b) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:49 and SEQ ID NO:51; (c) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO: 53 and SEQ ID NO:55; (d) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:57 and SEQ ID NO:59; (e) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:61 and SEQ ID NO:63; (f) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:65 and SEQ ID NO:67; (g) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:69 and SEQ ID NO:71; (h) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:73 and SEQ ID NO:75; (i) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:77 and SEQ ID NO:79; (j) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:81 and SEQ ID NO:83; and (k) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO:85 and SEQ ID NO:87; and (l) an antibody or antigen binding portion thereof comprising the variable domain amino acid sequences set forth in SEQ ID NO: 118 and SEQ ID NO:120. 51. A method of treating metabolic syndrome in a subject in need thereof comprising the step of administering to said subject an antibody or antigen-binding portion according to any one of paragraphs 1 to 25, wherein said antibody or antigen-binding portion inhibits myostatin activity. 52. A mammalian host cell line comprising polynucleotides encoding the heavy and light chains of a human, chimeric or humanized monoclonal antibody that competes for binding to myostatin with an antibody or an antigen-binding portion thereof, wherein the antibody or portion thereof has at least one property selected from the group consisting of: (a) competes for binding to myostatin with an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (b) binds to the same epitope of myostatin as an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (c) binds to myostatin with substantially the same KD as an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—1; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M; L-F58I, I85V; and (d) binds to myostatin with substantially the same off rate as an antibody selected from the group consisting of 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—1; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M; L-F58I, I85V. 53. A mammalian host cell line comprising polynucleotides encoding the heavy and light chains of a monoclonal antibody or antigen-binding portion thereof that competes for binding to myostatin with an antibody comprising: (a) a heavy chain that utilizes a human VH 1-02 gene, a human VH 3-21 gene or a human VH 3-23 gene; and (b) a light chain that utilizes a human VK L2 gene, a human VK A3 gene or a human VK A30 gene. 54. A mammalian host cell line comprising polynucleotides encoding the heavy and light chains of a monoclonal antibody or an antigen-binding portion of said monoclonal antibody having the same amino acid sequence as the antibody produced by a hybridoma cell line having ATCC Deposit Designation Number selected from the group consisting of PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576. 55. A mammalian host cell line comprising polynucleotides encoding an antibody having the same amino acid sequence as the antibody produced by a hybridoma cell having an ATCC Deposit Designation Number selected from the group consisting of PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576. 56. A method comprising expressing said human monoclonal antibody in said mammalian host cell line of any one of paragraphs 52-55 and recovering said human monoclonal antibody. 57. A hybridoma cell line selected from the group consisting of ATCC Deposit Designation Numbers PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576 58. A. mammalian host cell line comprising polynucleotides encoding the heavy and light chains of a human, chimeric or humanized monoclonal antibody, wherein the antibody or portion thereof has at least one property selected from the group consisting of: (a) competes for binding to myostatin with an antibody selected from the group consisting of: 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (b) binds to the same epitope of myostatin as an antibody selected from the group consisting of: 1—116—1; 1—136—3; 1—257—1; 1—46—1; 2—112—; 1—314—1; 1—66—1; 2—43—1; 2—177—1; 1—132—1; 1—268—1; 2—112_K; 1—116—1L-Q45K; 1—257—1L-L21I; 1—314—1H-T92A; 1—46—1H-L81M; 2—112—1H-I12V; 2—112—1L-F58I; 2—112—1L-I85V; 2—112—1H-L81M, L-F58I; 2—112—1H-L81M, L-I85V; and 2—112—1H-L81M, L-F58I, I85V; (c) is an antibody having the same amino acid sequence as the antibody produced by a hybridoma cell having an ATCC Deposit Designation Number selected from the group consisting of PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576; and (d) is a hybridoma cell line selected from the group consisting of ATCC Deposit Designation Numbers PTA-6566, PTA-6567, PTA-6568, PTA-6569, PTA-6570, PTA-6571, PTA-6572, PTA-6573, PTA-6574, PTA-6575, and PTA-6576.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a myostatin responsive reporter gene assay. As shown, neutralizing anti-myostatin antibodies inhibit myostatin induced luciferase activity in A204 cells. Human antibody variants 1—116—1L-Q45K; 1—257-1L-L21I; 1—314—1H-T92A and 2—112—1H-I12V, L-F 58I, I85V inhibited luciferase activity to the same extent as wild type antibodies.

FIG. 2 shows the results of an L6 Aurora beta-lactamase assay. As shown, neutralizing anti-myostatin antibodies inhibit myostatin induced beta-lactamase activity in L6 rat myoblasts. Human antibody variants 1—116—1L-Q45K; 1—257-1L-L21I; 1—314—1H-T92A and 2—112—1H-I12V, L-F58I, I85V inhibited beta-lactamase activity to the same extent as the wild type antibodies.

FIG. 3 shows the results of a western blot. As shown, neutralizing anti-myostatin antibodies of the invention inhibit myostatin induced smad2 phosphorylation in HepG2 cells by western blot.

FIG. 4 shows the results of myf5 mRNA expression assays. (A) Neutralizing anti-myostatin antibodies 1—268—1, 2—177—1, and 2—112—1 rescued myf5 gene expression inhibited by myostatin in C2C12 mouse myoblasts, whereas a non-neutralizing antibody 1—159—1 could not. Myf5 mRNA level was detected by TAQMAN® PCR. (B) 1—116—1 rescued myf5 gene expression in a dose-dependent manner.

FIG. 5 shows the results of C2C12 muscle cell differentiation assays.

(A) Neutralizing anti-myostatin antibodies 2—43—1, 1—314—1, and 1—257—1 rescued myostatin-blocked muscle differentiation in C2C12 mouse muscle cells. Embryonic myosin heavy chain (MHC) protein level was used a marker to measure C2C12 differentiation. (B) Antibodies 2—112—1, 1—46—1, and 1—116—1 rescued myostatin-blocked C2C12 muscle differentiation.

FIG. 6 (A) illustrates peptides generated from mature GDF8 (SEQ ID NO: 89) to test anti-myostatin antibody binding. Amino acids in lower case letters are the amino acids that are different from GDF11 (B) Summary of peptide binding. As shown in the Figure, some antibodies do not bind to any of the peptides. Some antibodies bind to non-contiguous peptides. (C) Predicted GDF8 structure with the illustration of peptide binding of the human anti-myostatin antibodies. GDF8 structure was generated using SWISS-MODEL; a fully automated protein structure homology-modeling server, accessible via the ExPASy web server, or from the program DeepView (Swiss Pdb-Viewer). The first seven amino acids were missing from the predicted structure. Mature GDF8 is a homodimer protein. Only one subunit is shown. The antibodies disclosed herein bind to GDF8 as a homodimer. As shown in the Figure, human monoclonal antibodies 2—43—1, 2—112—1, and 2—177—1 bind to both peptide 1 and 5. Peptides 1 and 5 are proximal spatially but not in primary structure.

FIG. 7 shows the amino acid sequences of peptides 1-11 and corresponding sequence identifiers (SEQ ID NOs 103-113).

FIG. 8 depicts epitope binning. Epitope binding of the human anti-myostatin antibodies of the invention was mapped by cross-competition experiments using a BIACORE® 3000 instrument. Antibodies are depicted as labeled boxes. Antibodies in one circle compete with antibodies in overlapping circles. For instance, antibody 1—46—1 competes with antibody 1—136—3, but antibody 1—46—1 does not compete with antibody 1—268—1. When two or more antibodies are in the same circle their respective binding cannot be distinguished.

FIG. 9 shows the results of an immunoprecipitation study to test the ability of human anti-myostatin antibodies of the invention to pull down mature GDF8 and mature GDF8/propeptide latent complex. Conditioned medium containing 293T cells transfected with GDF8 was used. As shown in the Figure, antibodies 2—112—1, 2—43—1, and 2—177—1 pulled down mature GDF8, mature GDF8/propeptide complex, and unprocessed GDF8; antibody 1—66—1 pulled down mature GDF8 and mature GDF8/propeptide complex; no other antibodies pulled down mature GDF8, propeptide, or unprocessed GDF8. Lane 4 is the 1/10 medium loading control, and lane 7 is immunoprecipitation control with medium only.

FIG. 10 shows the results of an immunoprecipitation study to test the ability of anti-myostatin antibodies of the invention to pull down mature GDF8 from mouse serum. As shown is the Figure, antibodies 2—112—1, 2—43—1, and 2—177—1 pulled down mature GDF8 from the mouse serum, whereas antibodies 1—116—1, and 1—66—1 could not.

FIG. 11 is a sequence alignment of mature human GDF8 and GDF11. Mature GDF8 (SEQ ID NO: 89) and GDF11 (SEQ ID NO: 90) share approximately 90% identical amino acid sequences. Both mature GDF8 and GDF11 form homodimers. Both mature GDF8 or GDF11 have nine cysteines, which form four internal disulfide bonds and one intermolecular disulfide bond. As shown in the Figure, cysteines that form a disulfide bond with each other are labeled with the same dots or plus. One cysteine (cys73) that forms intermolecular disulfide bond was labeled with a star. GDF8 structure was predicted using SWISS-MODEL (see FIG. 6C).

FIG. 12 is a table summarizing in vitro assay data.

FIG. 13 is a table summarizing gene usage, epitope binning and peptide binding.

FIGS. 14 and 15 show the effects on muscle mass in mice following in vivo treatment with anti-myostatin antibodies as compared with vehicle for the gastrocnemius-pantaris-sloeus (GPS), tibilalis anterior (TA) and quadriceps (Quads) muscles.

FIG. 16 shows the effects on muscle weight (A) and strength (B) in SCID mice following in vivo treatment with an anti-myostatin antibody (2—112_K).

FIG. 17 illustrates the effects on muscle weight (A; B; C and D) in SCID mice following in vivo treatment with varying doses of an anti-myostatin antibody (2—112_K).

FIG. 18 is a dose (A) and concentration (B) response analysis of varying doses of an anti-myostatin antibody (2—112_K) and their effects on muscle weight.

FIG. 19 is an alignment of the heavy (A) and light chain (B) variable regions of antibodies 2—112—1 (heavy chain: SEQ ID NO:77; light chain: SEQ ID NO:79) and 2—112_K (heavy chain: SEQ ID NO:118; light chain: SEQ ID NO:120).

FIG. 20 show the effects on muscle mass (A) and adipose mass (B) in mice following in vivo treatment with cortisone with an anti-myostatin antibody (2—112_K) as compared with vehicle for the gastrocnemius-pantaris-sloeus (GPS), tibilalis anterior (TA) and quadriceps (Quads) muscles (in 20A) and the inguinal, epididymal and abdominla fat pad masses (in 20B).

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer\'s specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.

The term “isolated protein”, “isolated polypeptide” or “isolated antibody” is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

In various embodiments an isolated anti-myostatin antibody is one that has been protein A-purified (see, e.g., Example XVI), one that has been synthesized by a hybridoma or other cell line in vitro, and/or a human anti-myostatin antibody derived from a transgenic mouse.

A protein or polypeptide is “substantially pure,” “substantially homogeneous,” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

The term “non-human transgenic organism” as used herein refers to any non-human transgenic individual living thing, including a non-human transgenic animal, plant, bacterium, protist, or fungus.

The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptide that comprises a segment that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to myostatin under suitable binding conditions, (2) ability to inhibit or activate myostatin. Typically, polypeptide analogs comprise a conservative amino acid substitution (or insertion or deletion) with respect to the native sequence. Analogs typically are at least 20 or 25 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full-length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analog antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from the germline amino acid sequence.

In certain embodiments, amino acid substitutions to an anti-myostatin antibody or antigen-binding portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogs, but still retain specific binding to myostatin. Analogs can include various muteins of a sequence other than the normally-occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made in the normally-occurring sequence, preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not alter the anti-parallel β-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. In general, glycine and proline would not be used in an anti-parallel β-sheet. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a desired biochemical property or pharmacological activity), such as a human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH-(cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO—, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

Where an “antibody” is referred to herein with respect to the invention, it is normally understood that an antigen-binding portion thereof may also be used. An antigen-binding portion competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

From N-terminus to C-terminus, both the mature light and heavy chain variable domains comprise, sequentially, the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain herein is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) or Chothia et al., Nature 342:878-883 (1989).

As used herein, an antibody that is referred to by number is the same as a monoclonal antibody that is obtained from the hybridoma of the same number. For example, monoclonal antibody 2—112 is the same antibody as one obtained from hybridoma 2—112, or a subclone thereof, such as 2—112—1, 2—112—2, and the like. The only exception is 1—136—3 which is a different hybridoma from subclones 1—136—1 and 1—136—2.

As used herein, a Fd fragment means an antibody fragment that consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546 (1989)) consists of a VH domain.

In some embodiments, the antibody is a single-chain antibody (scFv) in which VL and VH domains are paired to form a monovalent molecules via a synthetic linker that enables them to be made as a single protein chain. (Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988).) In some embodiments, the antibodies are diabodies, i.e., are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. (See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123 (1994).) In some embodiments, one or more CDRs from an antibody of the invention may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that specifically binds to myostatin. In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently. Further, the framework regions (FRs) may be derived from one of the anti-myostatin antibodies from which one or more of the CDRs are taken or from one or more different human antibodies.

In embodiments having one or more binding sites, the binding sites may be identical to one another or may be different.

As used herein, the term “human antibody” means any antibody in which the variable and constant domain sequences are human sequences. The term encompasses antibodies with sequences derived from (i.e., that utilize) human genes, but which have been changed, e.g. to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells. These antibodies may be prepared in a variety of ways, as described below.